Compounds active in sphingosine 1-phosphate signaling

ABSTRACT

The present invention relates to S1P analogs that have activity as S1P receptor modulating agents and the use of such compounds to treat diseases associated with inappropriate S1P receptor activity. The compounds have the general structure:
     structure:   

     
       
         
         
             
             
         
       
         
         
           
             wherein R 11  is C 5 -C 18  alkyl or C 5 -C 18  alkenyl; 
             Q is C 3 -C 6  optionally substituted cycloalkyl, C 3 -C 6  optionally substituted heterocyclic, C 3 -C 6  optionally substituted aryl C 3 -C 6  optionally substituted heteroaryl or —NH(CO)—; 
             R 3  is H, C 1 -C 4  alkyl, (C 1 -C 4  alkyl)OH or (C 1 -C 4  alkyl)NH 2 ; 
             R 23  is H or C 1 -C 4  alkyl, and 
             R 15  is hydroxy, phosphonate, or 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             wherein X and R 12  is O or S; 
             or a pharmaceutically acceptable salt or tautomer thereof.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/523,337, filed Jan. 28, 2005, now U.S. Pat. No. 7,241,790, whichis a national stage filing of International Patent Application Ser. No.PCT/US2003/023768 filed on 30 Jul. 2003, which claims priority from U.S.patent application Ser. No. 10/523,337, filed Jan. 28, 2005, U.S.Provisional Application Ser. No. 60/399,545, filed Jul. 30, 2002, andU.S. Provisional Application Ser. No. 60/425,595, filed Nov. 12, 2002,the disclosures of which are incorporated herein by reference.

US GOVERNMENT RIGHTS

This invention was made with United States Government support underGrant No. NIH R01 GM52722 and NIH R01 CA88994 awarded by NationalInstitutes of Health. The United States Government has certain rights inthe invention.

BACKGROUND

Sphingosine-1-phosphate (S1P) has been demonstrated to induce manycellular effects, including those that result in platelet aggregation,cell proliferation, cell morphology, tumor-cell invasion, endothelialcell chemotaxis and endothelial cell in vitro angiogenesis. For thesereasons, S1P receptors are good targets for therapeutic applicationssuch as wound healing and tumor growth inhibition.Sphingosine-1-phosphate signals cells in part via a set of Gprotein-coupled receptors named S1P1, S1P2, S1P3, S1P4, and S1P5(formerly Edg-1, Edg-5, Edg-3, Edg-6, and Edg-8, respectively). Thesereceptors share 50-55% identical amino acids and cluster with threeother receptors (LPA1, LPA2, and LPA3 (formerly Edg-2, Edg-4 and Edg-7))for the structurally-related lysophosphatidic acid (LPA).

A conformational shift is induced in the G-Protein Coupled Receptor(GPCR) when the ligand binds to that receptor, causing GDP to bereplaced by GTP on the α-subunit of the associated G-proteins andsubsequent release of the G-proteins into the cytoplasm. The α-subunitthen dissociates from the βγ-subunit and each subunit can then associatewith effector proteins, which activate second messengers leading to acellular response. Eventually the GTP on the G-proteins is hydrolyzed toGDP and the subunits of the G-proteins reassociate with each other andthen with the receptor. Amplification plays a major role in the generalGPCR pathway. The binding of one ligand to one receptor leads to theactivation of many G-proteins, each capable of associating with manyeffector proteins leading to an amplified cellular response.

S1P receptors make good drug targets because individual receptors areboth tissue and response specific. Tissue specificity of the S1Preceptors is desirable because development of an agonist or antagonistselective for one receptor localizes the cellular response to tissuescontaining that receptor, limiting unwanted side effects. Responsespecificity of the S1P receptors is also of importance because it allowsfor the development of agonists or antagonists that initiate or suppresscertain cellular responses without affecting other responses. Forexample, the response specificity of the S1P receptors could allow foran S1P mimetic that initiates platelet aggregation without affectingcell morphology.

Sphingosine-1-phosphate is formed as a metabolite of sphingosine in itsreaction with sphingosine kinase and is stored in abundance in theaggregates of platelets where high levels of sphingosine kinase existand sphingosine lyase is lacking. S1P is released during plateletaggregation, accumulates in serum and is also found in malignantascites. Biodegradation of S1P most likely proceeds via hydrolysis byectophosphohydrolases, specifically the sphingosine 1-phosphatephosphohydrolases.

The physiologic implications of stimulating individual S1P receptors arelargely unknown due in part to a lack of receptor type selectiveligands. Therefore there is a need for compounds that have strongaffinity and high selectivity for S1P receptor subtypes. Isolation andcharacterization of S1P analogs that have potent agonist or antagonistactivity for S1P receptors has been limited due to the complication ofsynthesis derived from the lack of solubility of S1P analogs. Thepresent invention is directed to a series of compounds that are activeat S1P receptors.

SUMMARY OF THE INVENTION

One embodiment of the present invention is directed to novelsphingosine-1-phosphate analogs, compositions comprising such analogs,and methods of using such analogs as agonist or antagonists ofsphingosine-1-phosphate receptor activity to treat a wide variety ofhuman disorders. S1P analogs of the present invention have a range ofactivities including agonism, with various degrees of selectivity atindividual S1P receptor subtypes, as well as compounds with antagonistactivity at the S1P receptors. More particularly, the S1P analogs of thepresent invention include compounds with the general structure:

-   -   wherein Q is selected from the group consisting of C₃-C₆        optionally substituted cycloalkyl, C₃-C₆ optionally substituted        heterocyclic, C₃-C₆ optionally substituted aryl, C₃-C₆        optionally substituted heteroaryl and

-   -   R₁ is selected from the group consisting of alkyl, alkenyl,        alkynyl, alkyl(optionally substituted aryl), arylalkyl and        arylalkyl(optionally substituted)aryl;    -   R₁₇ is H, alkyl, alkylaryl or alkyl(optionally substituted        aryl);    -   R₁₈ is N, O, S, CH or together with R₁₇ form a carbonyl group or        a bond;    -   W is NH, CH₂ or (CH₂)_(n)NH(CO);    -   R₂ and R₃ are independently selected from the group consisting        of H, NH₂, C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂,        with the proviso that R₂ and R₃ are not the same and either R₂        or R₃ is NH₂.    -   R₂₃ is selected from the group consisting of H, F, NH₂, OH,        CO₂H, C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;    -   R₂₄ is selected from the group consisting of H, F, CO₂H, OH and        PO₃H₂, or R₂₃ together with R₂₄ and the carbon to which they are        attached form a carbonyl group;    -   R₁₅ is selected from the group consisting of hydroxy,        phosphonate, and

-   -   wherein R₁₂ is selected from the group consisting of O, NH and        S;    -   X is selected from the group consisting of O, NH and S;    -   y is an integer ranging from 0-10; n is an integer ranging from        0-4; and

pharmaceutically acceptable salts and tautomers of such compounds, withthe proviso that R18 and W are not both CH2. Selective agonists andantagonists at S1P receptors will be useful therapeutically in a widevariety of human disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1F are graphic representations of [γ-35 S]GTP binding to HEK293Tcell membranes (containing different S1P receptors) in response to S1P,VPC23019 and VPC23031. FIG. 1A=S1P1 receptor, FIG. 1B=S1P3 receptor,FIG. 1C=S1P2 receptor, FIG. 1D=S1P4 receptor, FIG. 1E=S1P5 receptor, andFIG. 1F=S1P3 receptor. Each data point represents the mean of threedeterminations (CPM=counts per minute).

FIG. 2A-2E are graphic representations of [γ-35 S]GTP binding to HEK293Tcell membranes (containing different S1P receptors) in response to S1P,VPC23065 and VPC23069. FIG. 2A=S1P1 receptor, FIG. 2B=S1P3 receptor,FIG. 2C=S1P2 receptor, FIG. 2D=S1P4 receptor, and FIG. 2E=S1P5 receptor.Each data point represents the mean of three determinations (CPM=countsper minute).

FIG. 3A-3E are graphic representations of [γ-35 S]GTP binding to HEK293T cell membranes (containing different S1P receptors) in response toS1P, VPC23075 and VPC23079. FIG. 3A=S1P1 receptor, FIG. 3B=S1P3receptor, FIG. 3C=S1P2 receptor, FIG. 3D=S1P4 receptor, and FIG. 3E=S1P5receptor. Each data point represents the mean of three determinations(CPM=counts per minute).

FIG. 4A-4E are graphic representations of [γ-35 S]GTP binding to HEK293Tcell membranes (containing different S1P receptors) in response to S1P,VPC23087 and VPC23089. FIG. 4A=S1P1 receptor, FIG. 4B=S1P3 receptor,FIG. 4C=S1P2 receptor, FIG. 4D=S1P4 receptor, and FIG. 4E=S1P5 receptor.Each data point represents the mean of three determinations (CPM=countsper minute).

FIGS. 5A and 5B. FIG. 5A is a graphic representation of [γ-35 S]GTPbinding to HEK293T cell membranes containing the S1P1 receptor, inresponse to S1P, VPC23087 and VPC23087+S1P. FIG. 5B is a graphicrepresentation of [γ-35 S]GTP binding to HEK293T cell membranescontaining the S1P3 receptor, in response to S1P, VPC23089 andVPC23089+S1P. Each data point represents the mean of threedeterminations (CPM=counts per minute).

FIGS. 6A-6D are graphic representations of [γ-35 S]GTP binding toHEK293T cell membranes (containing different S1P receptors) in responseto S1P, VPC24289 and VPC24287. FIG. 6A=S1P1 receptor, FIG. 6B=S1P3receptor, FIG. 6C=S1P4 receptor, and FIG. 6D=S1P5 receptor. Each datapoint represents the mean of three determinations, wherein the activityof VPC24289 and VPC24287 is measured relative to S1P activity at thespecific receptor subtype.

DETAILED DESCRIPTION

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

As used herein, the term “purified” and like terms relate to anenrichment of a molecule or compound relative to other componentsnormally associated with the molecule or compound in a nativeenvironment. The term “purified” does not necessarily indicate thatcomplete purity of the particular molecule has been achieved during theprocess. A “highly purified” compound as used herein refers to acompound that is greater than 90% pure.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms.

As used herein, an “effective amount” means an amount sufficient toproduce a selected effect. For example, an effective amount of an S1Preceptor antagonist is an amount that decreases the cell signalingactivity of the S1P receptor.

As used herein, the term “halogen” or “halo” includes bromo, chloro,fluoro, and iodo.

The term “haloalkyl” as used herein refers to an alkyl radical bearingat least one halogen substituent, for example, chloromethyl, fluoroethylor trifluoromethyl and the like.

The term “C₁-C_(n) alkyl” wherein n is an integer, as used herein,represents a branched or linear alkyl group having from one to thespecified number of carbon atoms. Typically C₁-C₆ alkyl groups include,but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl,iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.

The term “C₂-C_(n) alkenyl” wherein n is an integer, as used herein,represents an olefinically unsaturated branched or linear group havingfrom 2 to the specified number of carbon atoms and at least one doublebond. Examples of such groups include, but are not limited to,1-propenyl, 2-propenyl, 1,3-butadienyl, 1-butenyl, hexenyl, pentenyl,and the like.

The term “C₂-C_(n) alkynyl” wherein n is an integer refers to anunsaturated branched or linear group having from 2 to the specifiednumber of carbon atoms and at least one triple bond. Examples of suchgroups include, but are not limited to, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 1-pentynyl, and the like.

The term “C₃-C_(n) cycloalkyl” wherein n=8, represents cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

As used herein, the term “optionally substituted” refers to from zero tofour substituents, wherein the substituents are each independentlyselected. Each of the independently selected substituents may be thesame or different than other substituents.

As used herein the term “aryl” refers to a mono- or bicyclic carbocyclicring system having one or two aromatic rings including, but not limitedto, phenyl, benzyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, andthe like. “Optionally substituted aryl” includes aryl compounds havingfrom zero to four substituents, and “substituted aryl” includes arylcompounds having one to three substituents, wherein the substituents,including alkyl, halo or amino substituents. The term (C₅-C₈ alkyl)arylrefers to any aryl group which is attached to the parent moiety via thealkyl group.

The term “heterocyclic group” refers to a mono- or bicyclic carbocyclicring system containing from one to three heteroatoms wherein theheteroatoms are selected from the group consisting of oxygen, sulfur,and nitrogen.

As used herein the term “heteroaryl” refers to a mono- or bicycliccarbocyclic ring system having one or two aromatic rings containing fromone to three heteroatoms and includes, but is not limited to, furyl,thienyl, pyridyl and the like.

The term “bicyclic” represents either an unsaturated or saturated stable7- to 12-membered bridged or fused bicyclic carbon ring. The bicyclicring may be attached at any carbon atom which affords a stablestructure. The term includes, but is not limited to, naphthyl,dicyclohexyl, dicyclohexenyl, and the like.

The term “lower alkyl” as used herein refers to branched or straightchain alkyl groups comprising one to eight carbon atoms, includingmethyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, neopentyl and thelike.

The terms 16:0, 18:0, 18:1, 20:4 or 22:6 hydrocarbon refers to abranched or straight alkyl or alkenyl group, wherein the first integerrepresents the total number of carbons in the group and the secondinteger represent the number of double bonds in the group.

As used herein, an “S1P modulating agent” refers a compound orcomposition that is capable of inducing a detectable change in S1Preceptor activity in vivo or in vitro (e.g., at least 10% increase ordecrease in S1P activity as measured by a given assay such as thebioassay described in Example 2).

As used herein, the term “EC₅₀ of an agent” refers to that concentrationof an agent at which a given activity, including binding of sphingosineor other ligand of an S1P receptor and/or a functional activity of a S1Preceptor (e.g., a signaling activity), is 50% maximal for that S1Preceptor. Stated differently, the EC₅₀ is the concentration of agentthat gives 50% activation, when 100% activation is set at the amount ofactivity of the S1P receptor which does not increase with the additionof more ligand/agonist and 0% is set at the amount of activity in theassay in the absence of added ligand/agonist.

As used herein, the term “phosphate analog” and “phosphonate analog”comprise analogs of phosphate and phosphonate wherein the phosphorousatom is in the +5 oxidation state and one or more of the oxygen atoms isreplaced with a non-oxygen moiety, including for example, the phosphateanalogs phosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate,phosphoramidate, boronophosphates, and the like, including associatedcounterions, e.g., H, NH₄, Na, and the like if such counterions arepresent.

The S1P analogs of the present invention contain one or more asymmetriccenters in the molecule. In accordance with the present invention astructure that does not designate the stereochemistry is to beunderstood as embracing all the various optical isomers, as well asracemic mixtures thereof.

The compounds of the present invention may exist in tautomeric forms andthe invention includes both mixtures and separate individual tautomers.For example the following structure:

is understood to represent a mixture of the structures:

The term “pharmaceutically-acceptable salt” refers to salts which retainthe biological effectiveness and properties of the S1P analogs of thepresent invention and which are not biologically or otherwiseundesirable. In many cases, the S1P analogs of the present invention arecapable of forming acid and/or base salts by virtue of the presence ofamino and/or carboxyl groups or groups similar thereto.

Pharmaceutically-acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines, such asalkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines,di(substituted alkenyl) amines, tri(substituted alkenyl) amines,cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines,substituted cycloalkyl amines, disubstituted cycloalkyl amine,trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines,disubstituted cycloalkenyl amine, trisubstituted cycloalkenyl amines,aryl amines, diaryl amines, triaryl amines, heteroaryl amines,diheteroaryl amines, triheteroaryl amines, heterocyclic amines,diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amineswhere at least two of the substituents on the amine are different andare selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic,and the like. Also included are amines where the two or threesubstituents, together with the amino nitrogen, form a heterocyclic orheteroaryl group. Examples of suitable amines include, by way of exampleonly, isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl)amine, tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol,tromethamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,N-alkylglucamines, theobromine, purines, piperazine, piperidine,morpholine, N-ethylpiperidine, and the like. It should also beunderstood that other carboxylic acid derivatives would be useful in thepractice of this invention, for example, carboxylic acid amides,including carboxamides, lower alkyl carboxamides, dialkyl carboxamides,and the like.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

EMBODIMENTS

One aspect of the present invention is directed to novel S1P analogsthat have activity as modulators of S1P receptor activity. Modulators ofS1P activity include agents that have either agonist or antagonistactivity at the S1P receptor as well as analogs of those compounds thathave been modified to resist enzymatic modification (i.e. blockmodification of the compounds by phosphohydrolases, sphingosine lyasesor sphingosine kinases), or provide a suitable substrate for sphingosinekinases to convert an administered form into a more active form.

The structure of S1P can be described as a combination of three regions:the phosphate head group, the linker region, and the fatty acid tail.Through structure activity relationships (SAR) of the closely relatedlysophospholipid, lysophosphatidic acid (LPA), it has been determinedthat the presence of a phosphate head group is an important feature toallow binding of S1P to its S1P receptors. However, there are exceptionsto the requirement for a phosphate head group. In particular aphosphonate, hydroxyl, phosphate or phosphonate group can be substitutedfor the phosphate head group while retaining activity at the S1Preceptor.

Based on the SAR of LPA, the linker region of S1P is anticipated to bethe area of the molecule that can best accommodate change. Again usingthe SAR of LPA as a lead, it is presumed that presence of a hydrogenbond donor 5 bonds away from the phosphate is important to binding. Froma retrosynthetic standpoint, the linker region may be seen as afunctionalized derivative of L-Serine.

Due to the long fatty acid chain and charged phosphate head group, S1Phas an amphipathic nature that makes it extremely insoluble in organicsolvents. Manipulation of the saturation of the fatty acid chain maycompromise aggregate formation of the molecule, thereby increasingsolubility. One important aspect of the long chain, however, is thelength. GTPγS studies that have been completed thus far havedemonstrated that an 18 carbon backbone, as is the case in S1P, displaysoptimal activity compared to 16 and 20 carbon backbones, however thelong fatty acid chain can vary from 8 to 25 carbons and still exhibitactivity.

It is also anticipated that the S stereochemistry of the C-2 amine mayhave an effect on binding as one would expect from a receptor. Hydrogenbonds from the phosphate head group and the C-2 amine to adjacentargenine and glutamic acid residues on the model receptor have beendemonstrated to be important to S1P-receptor binding. In accordance withone embodiment an S1P receptor modulating compound is provided whereinthe compound has the general structure:

-   -   wherein    -   W is CR₂₇R₂₈ or (CH₂)_(n)NH(CO);    -   wherein R₂₇ and R₂₈ are independently selected from the group        consisting of H, halo and hydroxy;    -   Y is selected from the group consisting of a bond, CR₉R₁₀,        carbonyl, NH, O or S;    -   wherein R₉ and R₁₀ are independently selected from the group        consisting of H, halo, hydroxy and amino;    -   Z is CH₂, aryl, flourine substituted aryl or heteroaryl;    -   R₁₁ and R₁₆ are independently selected from the group consisting        of C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, C₂-C₁₈ alkynyl, C₅-C₁₈ alkoxy,        (CH₂)_(p)O(CH₂)_(q), C₅-C₁₀ (aryl)R₂₀, C₅-C₁₀ (heteroaryl)R₂₀,        C₅-C₁₀ (cycloalkyl)R₂₀, C₅-C₁₀ alkoxy(aryl)R₂₀, C₅-C₁₀        alkoxy(heteroaryl)R₂₀ and C₅-C₁₀ alkoxy(cycloalkyl)R₂₀;    -   wherein R₂₀ is H or C₁-C₁₀ alkyl;    -   R₂₉ is H or halo;    -   R₁₇ is selected from the group consisting of H, halo, NH₂, C₁-C₆        alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₁-C₆ alkylcyano and        C₁-C₆ alkylthio;    -   R₂, and R₂₁ are both NH₂;    -   R₃ is selected from the group consisting of H, C₁-C₆ alkyl,        (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)aryl(C₀-C₄        alkyl) and (C₁-C₄ alkyl)aryloxyaryl(C₀-C₄ alkyl);    -   R₂₂ is selected from the group consisting of C₁-C₆ alkyl, (C₁-C₄        alkyl)OH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)aryl(C₀-C₄ alkyl) and        (C₁-C₄ alkyl)aryloxyaryl(C₀-C₄ alkyl);    -   R₂₃ is selected from the group consisting of H, F, NH₂, OH,        CO₂H, C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;    -   R₂₄ is selected from the group consisting of H, F, CO₂H, OH and        PO₃H₂, or R₂₃ together with R₂₄ and the carbon to which they are        attached form a carbonyl group;    -   R₂₅, R₇ and R₈ are independently selected from the group        consisting of O, S, CHR₂₆, CR₂₆, NR₂₆, and N;    -   wherein R₂₆ is H or C₁-C₄ alkyl;    -   R₁₅ is selected from the group consisting of hydroxy,        phosphonate, and

-   -   wherein R₁₂ is selected from the group consisting of O, NH and        S;    -   X is selected from the group consisting of O, NH and S;    -   y and m are integers independently ranging from 0 to 4;    -   p and q are integers independently ranging from 1 to 10;    -   n is an integer ranging from 0 to 10;    -   or a pharmaceutically acceptable salt or tautomer thereof, with        the proviso that W and Y are not both methylene.

In one embodiment, the present invention is directed to an S1P receptormodulating compound is represented by the formula:

wherein

-   -   Z is CH₂, aryl or heteroaryl;    -   R₁₆ is selected from the group consisting of C₁-C₁₈ alkyl,        C₂-C₁₈ alkenyl, C₂-C₁₈ alkynyl, C₅-C₁₈ alkoxy,        (CH₂)_(p)O(CH₂)_(q), C₅-C₁₀ (aryl)R₂₀, C₅-C₁₀ (heteroaryl)R₂₀,        C₅-C₁₀ (cycloalkyl)R₂₀, C₅-C₁₀ alkoxy(aryl)R₂₀, C₅-C₁₀        alkoxy(heteroaryl)R₂₀ and C₅-C₁₀ alkoxy(cycloalkyl)R₂₀, wherein        R₂₀ is H or C₁-C₁₀ alkyl;    -   R₁₇ is selected from the group consisting of H, halo, NH₂, C₁-C₆        alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₁-C₆ alkylcyano and        C₁-C₆ alkylthio;    -   R₂₁ is selected from the group consisting of NH₂, OH, C₁-C₆        alkyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)NH₂, (C₁-C₄        alkyl)aryl(C₀-C₄ alkyl) and (C₁-C₄ alkyl)aryloxyaryl(C₀-C₄        alkyl), with the proviso that R₂ or R₃ is NH₂;    -   R₁₅ is selected from the group consisting of hydroxy,        phosphonate, and

-   -   wherein X and R₁₂ are independently selected from the group        consisting of O and S;    -   R₂₃ is selected from the group consisting of H, F, NH₂, OH,        CO₂H, C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;    -   R₂₄ is selected from the group consisting of H, F, CO₂H, OH and        PO₃H₂, or R₂₃ together with R₂₄ and the carbon to which they are        attached form a carbonyl group; p and q are integers        independently ranging from 1 to 10;    -   y is an integer ranging from 0 to 4; and    -   n is an integer ranging from 0 to 10;    -   or a pharmaceutically acceptable salt or tautomer thereof. In        one embodiment the compound of Formula II is provided wherein Z        is CH₂, y is 0, n is 1-10, and R₁₇ is H. In another embodiment,        the compound of Formula II is provided wherein Z is C₅-C₆ aryl        or C₅-C₆ heteroaryl, y is 0, n is 0, R₁₇ and R₂₃ are each H and        R₁₆ is selected from the group consisting of C₅-C₁₂ alkyl,        C₂-C₁₂ alkenyl or C₅-C₁₂ alkoxy. In another embodiment, the        compound of Formula II is provided wherein Z is C₅-C₆ aryl or        C₅-C₆ heteroaryl, y is 0, n is 0, R₁₇, R₂₃ and R₂₄ are each H,        R₁₆ is selected from the group consisting of C₅-C₁₂ alkyl,        C₂-C₁₂ alkenyl or C₅-C₁₂ alkoxy and R₁₅ is hydroxy.

In another embodiment of the present invention, an S1P receptormodulating compound is provided wherein the compound is represented bythe formula:

-   -   wherein Z is aryl or heteroaryl;    -   R₁₆ is selected from the group consisting of C₅-C₁₈ alkyl,        C₅-C₁₈ alkenyl, C₅-C₁₈ alkynyl and C₅-C₁₈ alkoxy;    -   Y is selected from the group consisting of CHOH, CF₂, CFH,        carbonyl, NH, O and S;    -   W is CR₂₇R₂₈, wherein R₂₇ and R₂₈ are independently selected        from the group consisting of H, halo and hydroxy;    -   R₂₁ is selected from the group consisting of NH₂, OH, C₁-C₆        alkyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)NH₂, (C₁-C₄        alkyl)aryl(C₀-C₄ alkyl) and (C₁-C₄ alkyl)aryloxyaryl(C₀-C₄        alkyl);    -   R₂₃ is selected from the group consisting of H, F, NH₂, OH,        CO₂H, C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;    -   R₂₄ is selected from the group consisting of H, F, CO₂H, OH and        PO₃H₂, or R₂₃ together with R₂₄ and the carbon to which they are        attached form a carbonyl group;    -   R₁₅ is selected from the group consisting of hydroxy,        phosphonate, and

-   -   wherein X and R₁₂ are independently selected from the group        consisting of O and S; and y is an integer ranging from 0 to 4;    -   or a pharmaceutically acceptable salt or tautomer thereof. In        one embodiment the compound of Formula III is provided wherein Z        is C₅-C₆ aryl or C₅-C₆ heteroaryl, R₂₃ and R₂₄ are both H, R₂₁        is selected from the group consisting of OH, C₁-C₄ alkyl, and        (C₁-C₃ alkyl)OH; and y is 0.

In another embodiment, the compound is represented by the formula:

-   -   wherein R₁₆ is selected from the group consisting of C₅-C₁₂        alkyl, C₅-C₁₂ alkenyl and C₅-C₁₂ alkynyl;    -   Y is selected from the group consisting of carbonyl, NH and O;    -   R₁₅ is selected from the group consisting of hydroxy,        phosphonate, and

-   -   wherein X and R₁₂ are independently selected from the group        consisting of O and S;    -   R₂₁ is selected from the group consisting of C₁-C₆ alkyl, (C₁-C₄        alkyl)OH and (C₁-C₄ alkyl)NH₂;    -   R₂₃ and R₂₄ are independently selected from the group consisting        of H, OH, F, CO₂H or PO₃H₂ or R₂₃ together with R₂₄ and the        carbon to which they are attached form a carbonyl group, as well        as pharmaceutically acceptable salts and tautomers thereof.

In another embodiment, the compound of Formula III is provided wherein Zis C₅-C₆ aryl;

-   -   R₁₆ is selected from the group consisting of C₅-C₁₈ alkyl and        C₅-C₁₈ alkenyl;    -   Y is selected from the group consisting of CF₂, CFH, carbonyl,        NH, O and S;    -   W is CH₂;    -   R₂₁ is selected from the group consisting of C₁-C₆ alkyl, (C₁-C₄        alkyl)OH and (C₁-C₄ alkyl)NH₂;    -   R₂₃ and R₂₄ are both H; y is 0; and    -   R₁₅ is selected from the group consisting of hydroxy,        phosphonate, and

-   -   wherein R₁₂ is O and S (and in one embodiment R₁₅ is OH),    -   or a pharmaceutically acceptable salt or tautomer thereof.

In another embodiment of the present invention a S1P receptor modulatingcompound is provided wherein the compound is represented by the formula:

wherein

-   -   R₁₁ is selected from the group consisting of C₅-C₁₂ alkyl,        C₅-C₁₂ alkenyl and C₅-C₁₂ alkynyl;    -   R₂₉, is H or halo;    -   R₂₅, R₇ and R₈ are independently selected from the group        consisting of O, S, CHR₂₆, CR₂₆, NR₂₆, and N;    -   wherein R₂₆ is H, F or C₁-C₄ alkyl;    -   R₂, is selected from the group consisting of H, NH₂, OH, C₁-C₆        alkyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)NH₂, (C₁-C₄        alkyl)aryl(C₀-C₄ alkyl) and (C₁-C₄ alkyl)aryloxyaryl(C₀-C₄        alkyl);    -   R₁₅ is selected from the group consisting of hydroxy,        phosphonate, and

-   -   wherein X and R₁₂ are independently selected from the group        consisting of O and S;    -   R₂₃ and R₂₄ are independently selected from the group consisting        of H, OH, F, CO₂H, C₁-C₃ alkyl or PO₃H₂ or R₂₃ together with R₂₄        and the carbon to which they are attached form a carbonyl group;    -   m is 1 or 0; and    -   y is an integer ranging from 0 to 4;    -   or a pharmaceutically acceptable salt or tautomer thereof. In        one embodiment, R₂₉ is H or F; m is 0; y is 1 or 0; R₂ is        selected from the group consisting of H, C₁-C₆ alkyl and (C₁-C₄        alkyl)OH; R₂₄ is H and R₂₃ is C₁-C₃ alkyl. In accordance with        one embodiment of the present invention a compound of Formula        IV, V or VI is provided wherein R₂₃ and R₂₉ are both H; m is 0;        R₂₅ is CH₂ or CH; R₇ and R₈ are independently selected from the        group consisting of O, CH₂ or CH, NH, and N; R₂, is selected        from the group consisting of H, F, C₁-C₄ alkyl and (C₁-C₄        alkyl)OH; R₂₄ is selected from the group consisting of H, F,        C₁-C₃ alkyl; and y is 1 or 0.

In one embodiment of the present invention, an S1P receptor modulatingcompound is provided wherein the compound is represented by the formula:

wherein

-   -   R₁₁ is selected from the group consisting of C₅-C₁₈ alkyl,        C₅-C₁₈ alkenyl and C₅-C₁₈ alkynyl;    -   R₇ and R₈ are independently selected from the group consisting        of O, S, NH and N;    -   R₂, is selected from the group consisting of H, C₁-C₆ alkyl,        (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;    -   R₁₅ is selected from the group consisting of hydroxy,        phosphonate, and

-   -   wherein X and R₁₂ are independently selected from the group        consisting of O and S;    -   R₂₃ is selected from the group consisting of H, F and OH;    -   R₂₄ is selected from the group consisting of H, F, OH and PO₃H₂,        or R₂₃ together with R₂₄ and the carbon to which they are        attached form a carbonyl group;    -   m is 0; and    -   y is an integer ranging from 0 to 4;    -   or a pharmaceutically acceptable salt or tautomer thereof.

In one embodiment of the present invention a S1P receptor modulatingcompound is provided wherein the compound is represented by the formula:

-   -   wherein R₁₁ is selected from the group consisting of C₅-C₁₂        alkyl, C₅-C₁₂ alkenyl and C₅-C₁₂ alkynyl;    -   R₇ and R₈ are independently selected from the group consisting        of O, S, CH₂, CH, NH and N;    -   R₂ and R₃ are independently selected from the group consisting        of H, NH₂, OH, C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)NH₂,        (C₁-C₄ alkyl)aryl(C₀-C₄ alkyl) and (C₁-C₄        alkyl)aryloxyaryl(C₀-C₄ alkyl), with the proviso that R₂ and R₃        are not the same and either R₂ or R₃ is NH₂;    -   y is 1 or 0    -   R₁₅ is selected from the group consisting of hydroxy,        phosphonate, and

-   -   wherein R₁₂ is selected from the group consisting of O and S;    -   R₂₃ is selected from the group consisting of H, F, CO₂H, C₁-C₄        alkyl and OH;    -   R₂₄ is selected from the group consisting of H, F, C₁-C₄ alkyl        and PO₃H₂, or R₂₃ together with R₂₄ and the carbon to which they        are attached form a carbonyl group; as well as pharmaceutically        acceptable salts or tautomers thereof.

In accordance with one embodiment of the present invention, a compoundof Formula VII is provided wherein R₂₃ is H; R₂₄ is selected from thegroup consisting of H, F, C₁-C₄ alkyl; and R₇ and R₈ are independentlyselected from the group consisting of O, NH and N. In anotherembodiment, a compound of Formula VII is provided wherein R₂₃ is H; R₂is NH₂; and R₃ is selected from the group consisting of H, C₁-C₄ alkyl,(C₁-C₄ alkyl)OH and (C₁-C₄ alkyl)NH₂. Alternatively, in one embodiment acompound of Formula VII is provided wherein R₂₃ is H; R₃ is NH₂; and R₂is selected from the group consisting of H, C₁-C₄ alkyl, (C₁-C₄ alkyl)OHand (C₁-C₄ alkyl)NH₂. In another embodiment, a compound of Formula VIIis provided wherein R₂₃ is H; R₂ is NH₂; and R₃ is selected from thegroup consisting of H, C₁-C₄ alkyl, (C₁-C₄ alkyl)OH and (C₁-C₄alkyl)NH₂; R₂₄ is selected from the group consisting of H, F, C₁-C₄alkyl; and R₇ and R₈ are independently selected from the groupconsisting of O, NH and N. In another embodiment, a compound of FormulaVII is provided wherein R₁₁ is selected from the group consisting ofC₅-C₁₂ alkyl or C₅-C₁₂ alkenyl; R₇ and R₉ are independently selectedfrom the group consisting of O, NH and N; R₂ and R₃ are independentlyselected from the group consisting of H, NH₂, C₁-C₆ alkyl and (C₁-C₄alkyl)OH, with the proviso that R₂ and R₃ are not the same and either R₂or R₃ is NH₂; y is 0; R₁₅ is hydroxy; R₂₃ is H; and R₂₄ is H, F or C₁-C₄alkyl; as well as pharmaceutically acceptable salts or tautomersthereof.

In one embodiment of the present invention, a S1P receptor modulatingcompound is provided wherein the compound is represented by the formula:

-   -   wherein R₁₁ is selected from the group consisting of C₅-C₁₂        alkyl, C₅-C₁₂ alkenyl and C₅-C₁₂ alkynyl;    -   R₈ is O or N;    -   R₂ and R₃ are independently selected from the group consisting        of NH₂, C₁-C₆ alkyl and (C₁-C₄ alkyl)OH, with the proviso that        R₂ and R₃ are not the same and either R₂ or R₃ is NH₂;    -   R₁₅ is selected from the group consisting of hydroxy,        phosphonate, and

-   -   wherein R₁₂ is selected from the group consisting of O and S;    -   R₂₃ is H or F; and    -   R₂₄ is H, F or C₁-C₄ alkyl; as well as pharmaceutically        acceptable salts or tautomers thereof. In one embodiment the        compound of Formula VIII is provided wherein R₁₁ is C₅-C₁₂ alkyl        or C₅-C₁₂ alkenyl; R₈ is N; R₂ and R₃ are independently selected        from the group consisting of NH₂, CH₃ and (C₁-C₃ alkyl)OH, with        the proviso that R₂ and R₃ are not the same and either R₂ or R₃        is NH₂; and R₁₅ is hydroxy; R₂₃ is H; and R₂₄ is H or C₁-C₄        alkyl as well as pharmaceutically acceptable salts or tautomers        thereof. In another embodiment the compound of Formula VIII is        provided wherein R₁₁ is C₅-C₁₂ alkyl or C₅-C₁₂ alkenyl; R₈ is N;        R₂ and R₃ are independently selected from the group consisting        of NH₂, CH₃ and (C₁-C₃ alkyl)OH, with the proviso that R₂ and R₃        are not the same and either R₂ or R₃ is NH₂; and R₁₅ is hydroxy;        R₂₃ is H; and R₂₄ is H or CH₃ as well as pharmaceutically        acceptable salts or tautomers thereof.

In one embodiment, a S1P receptor modulating compound is providedwherein the compound is represented by the formula:

-   -   wherein R₁₁ is C₅-C₁₈ alkyl or C₅-C₁₈ alkenyl;    -   R₈ is N;    -   R₂ is NH₂;    -   R₃ is CH₃ or (C₁-C₃ alkyl)OH and R₁₅ is hydroxy;    -   or a pharmaceutically acceptable salt or tautomer thereof.

In accordance with one embodiment, an S1P receptor modulating compoundis provided wherein the compound has the general structure:

-   -   wherein R₁ is selected from the group consisting of alkyl,        alkenyl, alkynyl, alkyl(optionally substituted aryl),        alkyl(optionally substituted cycloalkyl), arylalkyl, and        arylalkyl(optionally substituted)aryl;    -   R₁₂ is 0, or R₁ and R₁₂ taken together form an optionally        substituted heteroaryl;    -   R₁₇ is H, C₁-C₄ alkyl or (CH₂)aryl;    -   R₂ and R₃ are independently selected from the group consisting        of H, NH₂, OH, C₁-C₆ alkyl, —(C₁-C₄ alkyl)OH, and —(C₁-C₄        alkyl)NH₂; y is an integer from 1-10, and R₄ is selected from        the group consisting of hydroxyl, phosphate, methylene        phosphonate, α-substituted methylene phosphonate, phosphate        analogs and phosphonate analogs or a pharmaceutically acceptable        salt thereof. In one embodiment one of the R₂ and R₃        substituents of Formula XII is NH₂. Examples of pharmaceutically        acceptable salts of the compounds of the Formula XII include        salts with inorganic acids, such as hydrochloride, hydrobromide        and sulfate, salts with organic acids, such as acetate,        fumarate, maleate, benzoate, citrate, malate, methanesulfonate        and benzenesulfonate salts, and when a carboxy group is present,        salts with metals such as sodium, potassium, calcium and        aluminium, salts with amines, such as triethylamine and salts        with dibasic amino acids, such as lysine. The compounds and        salts of the present invention encompass hydrate and solvate        forms.

In one embodiment, an S1P modulating compound is provided having thegeneral structure:

-   -   wherein R₁ is selected from the group consisting of C₈-C₂₂        alkyl, C₈-C₂₂ alkenyl, C₈-C₂₂ alkynyl and —(CH₂)_(n)-Z-R₆;    -   R₅ is selected from the group consisting of hydroxyl,        phosphonate, α-substituted methylene phosphonate, phosphate        analogs and phosphonate analogs;    -   y is an integer ranging from 1 to 4;    -   n is an integer ranging from 0 to 10;    -   Z is selected from the group consisting of cycloalkyl, aryl and        heteroaryl; and    -   R₆ is selected from the group consisting of H, C₁-C₁₂ alkyl,        C₁-C₂₀ alkoxy, C₁-C₂₀ alkylthio, and C₁-C₂₀ alkylamino or a        pharmaceutically acceptable salt thereof. When R₅ is an alpha        substituted phosphonate, the alpha carbon can be mono- or        di-substituted, wherein the substitutions are independently        selected from the group consisting of H, OH, F, CO₂H, PO₃H₂, or        together with the attached carbon, form a carbonyl. In one        embodiment, R₁ is C₈-C₂₂ alkyl, and more preferably C₁₂-C₁₆        alkyl, y is 1 or 2 and R₅ is hydroxy, phosphate or phosphonate.        Alternatively, in one embodiment, R₁ is —(CH₂)_(n)-Z-R₆, wherein        n is an integer ranging from 1-4, Z is aryl and R₆ is C₁-C₁₀        alkyl; more preferably, Z is phenyl, R₅ is hydroxy, phosphate or        phosphonate, and R₆ is C₆-C₁₀ alkyl.

In another embodiment of the present invention, an S1P modulatingcompound is provided having the general structure:

-   -   wherein R₁₄ is selected from the group consisting of H, hydroxy,        NH₂, C₈-C₂₂ alkyl, C₈-C₂₂ alkenyl, C₈-C₂₂ alkynyl and        —(CH₂)_(n)-Z-R₆;    -   R₄ is selected from the group consisting of hydroxyl, phosphate,        phosphonate, α-substituted methylene phosphonate, phosphate        analogs and phosphonate analogs;    -   y is an integer ranging from 1 to 4;    -   m is an integer ranging from 0 to 4;    -   n is an integer ranging from 0 to 10;    -   Z is selected from the group consisting of cycloalkyl, aryl and        heteroaryl; and    -   R₆ is selected from the group consisting of H, C₁-C₁₂ alkyl,        C₁-C₂₀ alkoxy, C₁-C₂₀ alkylthio, and C₁-C₂₀ alkylamino; and    -   R₇ and R₈ are independently selected from the group consisting        of O, S and N. In one embodiment R₁ is selected from the group        consisting of C₈-C₂₂ alkyl, C₈-C₂₂ alkenyl and C₈-C₂₂ alkynyl,        R₄ is hydroxyl, phosphate or phosphonate, y is 1 or 2, m is 0 or        1 and either R₇ or R₈ is N; more preferably, R₁ is C₄-C₁₀ alkyl,        R₄ is hydroxyl or phosphate, y is 1, m is 0 and R₇ and R₈ are        both N.

The present invention also encompasses compounds of the generalstructure:

-   -   wherein R₉ is selected from the group consisting of —NR₁, and        —OR₁;    -   R₁ is selected from the group consisting of C₈-C₂₂ alkyl and

-   -   wherein R₆ and R₁₃ are independently selected from the group        consisting of H, C₁-C₁₀ alkyl and C₁-C₂₀ alkoxy and R₁₀ is        hydroxy, phosphonate, methylene phosphonate or phosphate, with        the proviso that when R₉ is —NR₁, R₁₀ is not phosphate. In one        preferred embodiment, R₉ is —NR₁, R₆ is C₁-C₁₀ alkyl, R₁₃ is H        and R₁₀ is hydroxy, phosphonate, or methylene phosphonate.

A GTP[γ35 S] binding assay was developed to analyze directly theactivation of individual S1P receptors, and thus allow theidentification of S1P receptor agonists and antagonists as well asdetermine the relative efficacies and potencies at each receptor in acommon system. The same results were obtained regardless of whether therecombinant receptor used exogenous G proteins (HEK293T cells) orendogenous G proteins (RH7777 cells). In addition, insect Sf9 cellsinfected with recombinant baculovirus encoding receptors (e.g. LPA andS1P receptors) and G proteins can also serve as the source of membranesfor the broken cells used in the GTPgammaS-35 binding assays. The Sf9cell and HEK293T cell membranes gave similar results. Furthermore, theactivities measured in the broken cell assay predicted the responsesseen in whole cell assays. Thus the primary assay used in the presentinvention for determining compound potency and efficacy is a validmeasure of activity at the S1P receptors.

The GTP[γ35 S] binding assay has revealed that the compounds of thepresent invention have the ability to modulate S1P receptor activity(See Examples 2 and 3). More particularly, compounds represented by thefollowing formula display activity as modulators of S1P activity. Moreparticularly, such compounds include those having the structure

-   -   wherein    -   W is CR₂₇R₂₈ or (CH₂)_(n)NH(CO);    -   wherein R₂₇ and R₂₈ are independently selected from the group        consisting of H, halo and hydroxy;    -   Y is selected from the group consisting of a bond, CR₉R₁₀,        carbonyl, NH, O or S;    -   wherein R₉ and R₁₀ are independently selected from the group        consisting of H, halo, hydroxy and amino;    -   Z is CH₂, aryl, halo substituted aryl or heteroaryl;    -   R₁₁ and R₁₆ are independently selected from the group consisting        of C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, C₂-C₁₈ alkynyl, C₅-C₁₈ alkoxy,        (CH₂)_(p)O(CH₂)_(q), C₅-C₁₀ (aryl)R₂₀, C₅-C₁₀ (heteroaryl)R₂₀,        C₅-C₁₀ (cycloalkyl)R₂₀, C₅-C₁₀ alkoxy(aryl)R₂₀, C₅-C₁₀        alkoxy(heteroaryl)R₂₀ and C₅-C₁₀ alkoxy(cycloalkyl)R₂₀;    -   wherein R₂₀ is H or C₁-C₁₀ alkyl;    -   R₂₉ is H or halo;    -   R₁₇ is selected from the group consisting of H, halo, NH₂, C₁-C₆        alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₁-C₆ alkylcyano and        C₁-C₆ alkylthio;    -   R₂ and R₂₁ are both NH₂;    -   R₃ is selected from the group consisting of H, C₁-C₆ alkyl,        (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;    -   R₂₂ is selected from the group consisting of C₁-C₆ alkyl, (C₁-C₄        alkyl)OH and (C₁-C₄ alkyl)NH₂;    -   R₂₃ is selected from the group consisting of H, F, CO₂H, OH,        C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;    -   R₂₄ is selected from the group consisting of H, F and PO₃H₂, or        R₂₃ together with R₂₄ and the carbon to which they are attached        form a carbonyl group;    -   R₂₅, R₇ and R₈ are independently selected from the group        consisting of O, S, CHR₂₆, CHR₂₆, NR₂₆, and N;    -   wherein R₂₆ is H, F or C₁-C₄ alkyl;    -   R₁₅ is selected from the group consisting of hydroxy,        phosphonate, and

-   -   wherein R₁₂ is selected from the group consisting of O, NH and        S;    -   X is selected from the group consisting of O, NH and S;    -   y and m are integers independently ranging from 0 to 4;    -   p and q are integers independently ranging from 1 to 10;    -   n is an integer ranging from 0 to 10;    -   or a pharmaceutically acceptable salt or tautomer thereof, with        the proviso that W and Y are not both methylene.

As described in Example 2 compounds having the general structure

-   -   wherein R₉ is selected from the group consisting of —NR₁, and        —OR₁, R₁ is C₈-C₂₂ alkyl, R₂ and R₃ are independently selected        from the group consisting of H and NH₂, wherein at least one of        R₂ and R₃ is NH₂ and R₄ is phosphate all display significant        agonist activity at the S1P receptors tested (S1P1, S1P2, S1P3,        S1P5), although none were as potent as S1P itself (See Table 1        of Example 2). However, one compound, VPC22135 (wherein R₂ is H,        R₃ is NH₂, R₄ is phosphate and R₉ is —N(CH₂)₁₃CH₃), approached        the potency of S1P at both the human S1P1 and human S1P3        receptors. In accordance with one embodiment of the present        invention, compound VPC22135 is used as a selective agonist of        human S1P1 and human S1P3 receptors. Curiously, this compound        has the amino group in the unnatural (R) configuration. Its        enantiomer, VPC22053, was more than 1 log order less potent at        both the S1P1 and S1P3 receptors.

An additional series of compounds have shown activity in modulating S1Preceptor activity, however these compounds also displayed selectivityfor certain S1P receptor subtypes (See Example 3 and FIGS. 1-5). Each ofthese compounds (VPC 23019, 23031, 23065, 23069, 23087, 23089, 23075,23079) are inactive at the S1P2 receptor. Compounds VPC23031, 23019,23089 are inverse agonists (antagonists of the S1P3) receptor, but thisinverse agonism becomes agonism when the alkyl chain length is 9 carbons(VPC23079) or 10 (VPC23069). In accordance with one embodiment of thepresent invention an antagonist of S1P activity is provided. Inparticular, a compound having the general structure:

-   -   wherein R₁ and R₁₁ is C₄-C₁₂ alkyl and located in the meta or        ortho position,    -   Q is selected from the group consisting of C₃-C₆ optionally        substituted cycloalkyl, C₃-C₆ optionally substituted        heterocyclic, C₃-C₆ optionally substituted aryl and C₃-C₆        optionally substituted heteroaryl;    -   R₃ is selected from the group consisting of H, C₁-C₄ alkyl and        (C₁-C₄ alkyl)OH;    -   R₂₃ is selected from the group consisting of H, F, CO₂H, OH,        C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;    -   R₂₄ is selected from the group consisting of H, F and PO₃H₂, or        R₂₃ together with R₂₄ and the carbon to which they are attached        form a carbonyl group; and    -   R₁₅ is selected from the group consisting of hydroxy,        phosphonate, and

-   -   wherein X and R₁₂ is selected from the group consisting of O and        S;    -   or a pharmaceutically acceptable salt or tautomer thereof are        anticipated to have antagonist activity at the S1P3 receptor. In        accordance with one embodiment, the R₁ substituent is located in        the ortho position on the phenyl ring, and in one embodiment,        the R₁ substituent is located in the meta position on the phenyl        ring.

However compounds of the general structure

(wherein R₁₁ is located in the para-position) have exhibited activity asagonists of S1P activity. In particular compounds of Formula XI areprovided as S1P agonists wherein R₁₁ is C₅-C₁₈ alkyl or C₅-C₁₈ alkenyl;

-   -   Q is selected from the group consisting of C₃-C₆ optionally        substituted cycloalkyl, C₃-C₆ optionally substituted        heterocyclic, C₃-C₆ optionally substituted aryl and C₃-C₆        optionally substituted heteroaryl;    -   R₃ is selected from the group consisting of H, C₁-C₄ alkyl and        (C₁-C₄ alkyl)OH;    -   R₂₃ is selected from the group consisting of H, F, CO₂H, OH,        C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;    -   R₂₄ is selected from the group consisting of H, F and PO₃H₂, or        R₂₃ together with R₂₄ and the carbon to which they are attached        form a carbonyl group; and    -   R₁₅ is selected from the group consisting of hydroxy,        phosphonate, and

-   -   wherein X and R₁₂ are independently selected from the group        consisting of O and S;    -   or a pharmaceutically acceptable salt or tautomer thereof and a        pharmaceutically acceptable carrier. In one embodiment, a        compound represented by Formula XI is provided as an S1P agonist        wherein    -   R₁₁ is C₅-C₁₈ alkyl or C₅-C₁₈ alkenyl;    -   Q is —NH(CO)—,

-   -   R₃ is selected from the group consisting of H, C₁-C₄ alkyl and        (C₁-C₄ alkyl)OH;    -   R₂₄ is H;    -   R₂₃ is H or C₁-C₄ alkyl, and    -   R₁₅ is selected from the group consisting of hydroxy,        phosphonate, and

-   -   wherein X and R₁₂ are independently selected from the group        consisting of O and S.

Compounds VPC23065, VPC23087 and VPC23075 are primary alcohols, i.e. R₄of formula XII is hydroxy. These compounds demonstrate significantagonist activity at various S1P receptors. In particular, the S1P4receptor binds to the primary alcohol S1P analogs with an EC₅₀ within alog order of the phosphorylated compounds. Since S1P4 is present onlymphocytes, the use of the primary alcohol analogs may be used forimmuno-suppression. In addition, it is also hypothesized that thehydroxy moiety of the primary alcohols may be converted to phosphates invivo. Therefore the primary alcohol S1P analogs of the present inventionare all anticipated to serve as prodrug forms of active S1P receptormodulating compounds.

S1P is metabolized by a variety of conceivable routes includingphosphatases, esterases or transported into cells. The S1P signal atreceptors might be prolonged if the routes of degradation could beevaded or inhibited by S1P structural analogs. The S1P analogs of thepresent invention can be used, in accordance with one embodiment, toinhibit or evade endogenous S1P metabolic pathways includingphosphotases, esterases, transporters and S1P acyl transferases. Forexample, those S1P analogs that lack an ester bond would be resistant todegradation by endogenous esterases. One embodiment of the presentinvention is directed to compounds that function as a S1P receptoragonists and antagonists that are resistant to hydrolysis by lipidphosphate phosphatases (LPPs) or are sub-type selective inhibitors ofLPPs, and in particular are resistant to hydrolysis by sphingosine1-phosphate phosphohydrolase. Previously described S1P mimetics containa phosphate group, and thus are likely susceptible to hydrolysis byLPPs.

Alpha hydroxy phosphonates are well known phosphate mimetics. Forexample, the compounds used clinically to treat osteoporosis(pamidronate, alendronate) are alpha hydroxy bisphosphonates that areanalogs of pyrophosphate. S1P analogs can be prepared wherein thephosphate moiety is replaced by an alpha substituted phosphonate,wherein the substituents are selected from the group consisting of H,OH, F, CO₂H, PO₃H₂ or double bonded oxygen. Accordingly, one aspect ofthe present invention is directed to lipid phosphate phosphataseresistant S1P analogs having the general structures:

-   -   wherein R₉ is selected from the group consisting of —NR₁, and        —OR₁;    -   R₁ is selected from the group consisting of C₈-C₂₂ alkyl, C₈-C₂₂        alkenyl, C₈-C₂₂ alkynyl and —(CH₂)_(n)-Z-R₆;    -   R₁₁ is —(CH₂)_(n)-Z-R₆; wherein n is an integer ranging from 0        to 10, Z is selected from the group consisting of aryl and        heteroaryl and R₆ is selected from the group consisting of H,        C₁-C₁₀ alkyl, C₁-C₂₀ alkoxy, C₁-C₂₀ alkylthio, and C₁-C₂₀        alkylamino;    -   R₂ and R₃ are independently selected from the group consisting        of H, NH₂, OH, C₁-C₆ alkyl, —(C₁-C₄ alkyl)OH, —(C₁-C₄ alkyl)NH₂,        —(C₁-C₄ alkyl)aryl(C₀-C₄ alkyl) and —(C₁-C₄        alkyl)aryloxyaryl(C₀-C₄ alkyl), wherein R₂ and R₃ are not the        same and R₂ or R₃ is NH₂    -   y is an integer from 0-10;    -   R₁₄ is selected from the group consisting of

-   -   R₁₅ is selected from the group consisting of H, hydroxy, amino,        COOH, halo, PO₂H₂; or R₁₅ and R₁₆ taken together form a keto        group or a methylene group;    -   R₁₆ is selected from the group consisting of hydroxy, amino,        COOH, halo,    -   PO₂H₂; or R₁₅ and R₁₆ taken together with the carbon to which        they are bound form a carbonyl or a methylene group; and    -   R₁₇ is selected from the group consisting of O, S and NH. In one        preferred embodiment, R₉ is —NR₁, wherein R₁ is C₈-C₂₂ alkyl or        —(CH₂)_(n)-Z-R₆, y is 0 or 1, R₁₅ and R₁₆ are independently H,        C₁-C₄ alkyl or hydroxyl, and R₁₄ is OH. In an alternative        preferred embodiment, the compound has the general structure:

-   -   wherein R₉ is selected from the group consisting of —NR₁, and        —OR₁;    -   R₁ is selected from the group consisting of C₈-C₂₂ alkyl, C₈-C₂₂        alkenyl, C₈-C₂₂ alkynyl and —(CH₂)_(n)-Z-R₆, wherein n is an        integer ranging from 0 to 10, Z is selected from the group        consisting of aryl and heteroaryl and R₆ is selected from the        group consisting of H, C₁-C₁₀ alkyl, C₁-C₂₀ alkoxy, C₁-C₂₀        alkylthio, and C₁-C₂₀ alkylamino;    -   R₂ is NH₂ or OH;    -   y is an integer from 0-10;    -   R₁₄ is H or

-   -   R₁₅ is NH₂ or OH; and    -   R₁₇ is selected from the group consisting of O, S and NH. In one        preferred embodiment, R₉ is —NR₁, wherein R₁ is C₈-C₂₂ alkyl or        —(CH₂)_(n)-Z-R₆, y is 0 or 1, and R₁₇ is O.

Lysophospholipids such as S1P and LPA, and their phosphate-containinganalogs, are probably degraded by membrane bound lipidectophosphohydrolases. This activity can be evaded by substitutingphosphonate, α-substituted phosphonate, phosphothionate or otherphosphate analogs as phosphate surrogates. Such compounds might alsofunction as lipid ectophosphohydrolase inhibitors. Further, substitutionof small alkyl groups (e.g. C₁-C₄ alkyl, C₁-C₃ alkylalcohol) at C-1 orC-2 might retard lipid ectophosphohydrolase cleavage by sterichindrance.

In accordance with one embodiment, an S1P receptor modulating compoundis provided wherein the compound has the general structure:

-   -   wherein R₁ is selected from the group consisting of alkyl,        alkenyl, alkynyl, alkyl (optionally substituted aryl), alkyl        (optionally substituted cycloalkyl), arylalkyl and arylalkyl        (optionally substituted aryl) R₇ is H, O, or R₁ and R₇ taken        together form an optionally substituted C₃-C₆ heteroaryl or        optionally substituted C₃-C₆ heterocyclic group; R₆ is H, C₁-C₄        alkyl or (CH₂)aryl; R₂ and R₃ are independently selected from        the group consisting of H, NH₂, OH, C₁-C₆ alkyl, —(C₁-C₄        alkyl)OH, and —(C₁-C₄ alkyl)NH₂; R₄ and R₅ are independently        selected from the group consisting of H, NH₂, OH, C₁-C₆ alkyl,        —(C₁-C₄ alkyl)OH, and —(C₁-C₄ alkyl)NH₂; R₈ is O, NH or S. In        one embodiment, one of the R₂ and R₃ substituents is NH₂ while        the other is CH₃ and R₆ is H. In another embodiment, one of the        R₂ and R₃ substituents is NH₂ while the other is H and one of        the R₄ and R₅ substituents is CH₃ while the other is H, and R₆        is H.

In accordance with one embodiment of the invention, a compound isprovided that could be converted by phosphorylation to an S1P receptormodulating compound. The compound has the general structure:

-   -   wherein R₁ is selected from the group consisting of alkyl,        alkenyl, alkynyl, alkyl (optionally substituted aryl), alkyl        (optionally substituted cycloalkyl), arylalkyl and arylalkyl        (optionally substituted aryl) R₇ is H, O, or R₁ and R₇ taken        together form an optionally substituted C₃-C₆ heteroaryl or        optionally substituted C₃-C₆ heterocyclic group; R₆ is H, C₁-C₄        alkyl or (CH₂)aryl; R₂ and R₃ are independently selected from        the group consisting of H, NH₂, OH, C₁-C₆ alkyl, —(C₁-C₄        alkyl)OH, and —(C₁-C₄ alkyl)NH₂; R₄ and R₅ are independently        selected from the group consisting of H, NH₂, OH, C₁-C₆ alkyl,        —(C₁-C₄ alkyl)OH, and —(C₁-C₄ alkyl)NH₂. In one embodiment, one        of the R₂ and R₃ substituents is NH₂ while the other is CH₃ and        R₆ is H. In another embodiment, one of the R₂ and R₃        substituents is NH₂ while the other is H and one of the R₄ and        R₅ substituents is CH₃ while the other is H, and R₆ is H.

In accordance with one embodiment, an S1P receptor modulating compoundis provided wherein the compound has the general structure:

-   -   wherein R₁ is selected from the group consisting of C₁-C₆ alkyl,        C₁-C₆ alkenyl, C₁-C₆ alkynyl, or C₁-C₆ alkyl, C₁-C₆ alkenyl,        C₁-C₆ alkynyl optionally substituted with OH;    -   R₂ is C₅-C₁₀ alkyl, C₅-C₁₀ alkoxy, (CH₂)_(n)O(CH₂)_(m), C₅-C₁₀        (optionally substituted aryl), C₅-C₁₀ (optionally substituted        heteroaryl), C₅-C₁₀ (optionally substituted cycloalkyl), C₅-C₁₀        alkoxy (optionally substituted aryl), C₅-C₁₀ alkoxy (optionally        substituted heteroaryl) and C₅-C₁₀ alkoxy (optionally        substituted cycloalkyl);    -   R₃ is selected from the group consisting of H, halo, C₁-C₆        alkoxy, C₁-C₆ alkyl, (CH₂)_(y)NH₂, (CH₂)_(y)cyano and C₁-C₆        alkylthio;    -   R₄ is selected from the group consisting of hydroxy, phosphate,        methylene phosphonate, α-substituted methylene phosphonate,        thiophosphate, thiophosphonate and other phosphate analogs and        phosphonate analogs or a pharmaceutically acceptable salt        thereof;    -   R₅ is selected from the group consisting of H, halo, C₁-C₄ alkyl        and haloalkyl;    -   X is CR₈R₉;    -   Y is selected from the group consisting of CR₈R₉, carbonyl, NH,        O or S;    -   R₈ and R₉ are independently selected from the group consisting        of H, halo and hydroxy;    -   n and m are integers independently ranging from 5-10, and    -   y is an integer ranging from 0-10 with the proviso that X and Y        are not both methylene. In one embodiment, a compound of the        Formula IX is provided wherein R₅ is selected from the group        consisting of H, F, methyl and ethyl. In another embodiment, a        compound of the Formula IX is provided wherein X is selected        from the group consisting of CH₂, CHF, CF₂, and CHOH. In a        further embodiment, a compound of the Formula IX is provided        wherein    -   R₁ is selected from the group consisting of CH₃, CH₂CH₃, CH₂OH,        CH₂CH₂OH and CH₂CH₂CH₂OH;    -   R₂ is C₅-C₁₀ alkyl, C₅-C₁₀ alkoxy, (CH₂)_(n)O(CH₂)_(m), C₅-C₁₀        (optionally substituted aryl), C₅-C₁₀ (optionally substituted        heteroaryl) and C₅-C₁₀ (optionally substituted cycloalkyl);    -   R₃ and R₅ are H;    -   R₄ is selected from the group consisting of hydroxy, phosphate        and methylene phosphonate;    -   X is CH₂;    -   Y is selected from the group consisting of carbonyl, NH, O and        S; and    -   n and m are integers independently ranging from 5-10. In one        embodiment a compound of Formula IX is provided wherein R₁ is        —CH₃, or —CH₂CH₃; R₂ is C₅-C₁₀ alkyl; R₃ and R₅ are H; R₄ is        hydroxy or phosphate X is CH₂; and Y is selected from the group        consisting of carbonyl, NH and O.

The present invention also encompasses the pharmaceutically acceptablesalts of the compounds of the Formula IX including salts with inorganicacids, such as hydrochloride, hydrobromide and sulfate, salts withorganic acids, such as acetate, fumarate, maleate, benzoate, citrate,malate, methanesulfonate and benzenesulfonate salts, and when a carboxygroup is present, salts with metals such as sodium, potassium, calciumand aluminium, salts with amines, such as triethylamine and salts withdibasic amino acids, such as lysine. The compounds and salts of thepresent invention encompass hydrate and solvate forms.

In one embodiment, an S1P modulating compound is provided having thegeneral structure:

-   -   wherein R₁ is methyl or ethyl;    -   R₂ is selected from the group consisting of C₅-C₁₀ alkyl,        (CH₂)_(n)O(CH₂)_(m),    -   C₅-C₁₀ (optionally substituted aryl), C₅-C₁₀ (optionally        substituted heteroaryl), C₅-C₁₀ (optionally substituted        cycloalkyl), C₅-C₁₀ alkoxy (optionally substituted aryl), C₅-C₁₀        alkoxy (optionally substituted heteroaryl) and C₅-C₁₀ alkoxy        (optionally substituted cycloalkyl);    -   R₄ is OPO₃H₂ or OH;    -   n and m are integers independently ranging from 0 to 10;    -   X is a methylene group optionally substituted with one or two        fluorine atoms or a secondary alcohol in either        stereoconfiguration;    -   Y is a carbonyl group, —O—, —NH— or a methylene group that is        optionally substituted with one or two fluorine atoms, or a        secondary alcohol in either stereoconfiguration, with the        proviso that X and Y are not both methylene. In one embodiment,        the compound of Formula X is provided wherein R₁ is methyl or        ethyl; R₂ is C₅-C₁₀ alkyl or (CH₂)_(n)O(CH₂)_(m); R₄ is OPO₃H₂        or OH; X is methylene; Y is a carbonyl group, —O— or —NH—; and n        and m are integers independently ranging from 0 to 10. More        particularly, in one embodiment, compounds of Formula X are        provided wherein R₁ is methyl; R₂ is C₅-C₈ alkyl and located in        the para position; R₄ is OPO₃H₂ or OH; X is methylene; and Y is        a carbonyl group or —NH—.

In accordance with one embodiment, compounds suitable for use inaccordance with the present invention include:

-   -   wherein R₁ is selected from the group consisting of —CH₃,        —CH₂CH₃, CH₂OH, CH₂CH₂OH; R₃ is selected from the group        consisting of H, C₁-C₆ alkoxy and C₁-C₆ alkyl; Y is selected        from the group consisting of CHOH, CF₂, CFH, carbonyl, NH, O and        S; and R₁₂ is H, C₁-C₆ alkoxy or C₁-C₆ alkyl. More particularly,        suitable compounds include the following compounds:

The present invention also encompasses compounds general structure:

-   -   wherein R₁ and R₁ are independently selected from the group        consisting of C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl;    -   R_(1g) is selected from the group consisting of C₁-C₆ alkyl and        (C₁-C₆ alkyl)OH;        Q is

-   -   R₂ is C₅-C₁₂ alkyl, C₂-C₁₂ alkenyl (CH₂)_(n)O(CH₂)_(m), C₅-C₁₀        (optionally substituted aryl),    -   C₅-C₁₀ (optionally substituted heteroaryl) and C₅-C₁₀        (optionally substituted cycloalkyl);    -   R₃ is selected from the group consisting of H, halo, C₁-C₆        alkoxy, C₁-C₆ alkyl, (CH₂)_(n)NH₂, (CH₂), cyano and C₁-C₆        alkylthio;    -   R₄ is selected from the group consisting of hydroxy,

-   -   R₅ is selected from the group consisting of H, F, methyl or        ethyl;    -   X is CH₂, CHF, CF₂ or CHOH;    -   Y is selected from the group consisting of CHF, CF₂, CHOH,        carbonyl, NH, O or S;    -   n and m are integers independently ranging from 0-10, with the        proviso that X and Y are not both methylene. In one embodiment,        R₁ is methyl or ethyl, R₂ is C₅-C₁₀ alkyl, C₅-C₁₀ aryl or C₅-C₁₀        alkoxy, R₃ is H, C₁-C₆ alkoxy or C₁-C₆ alkyl, R₄ is as defined        immediately above, R₅ is H, X is methylene and Y is a carbonyl        group, —O— or —NH—; or a pharmaceutically acceptable salt or        tautomer thereof. In another embodiment, Q is

-   -   R₂ and R₁ are independently selected from the group consisting        of C₅-C₁₂ alkyl and C₂-C₁₂ alkenyl and R₁₅ is OH.

The compounds of the present invention are anticipated to be highaffinity agonists (or antagonists) at various sphingosine I-phosphatereceptors of the ‘Edg’ family. The compounds of the present inventionare also expected to evoke lymphopenia when introduced into rodents orhumans. Thus the compounds of the invention are immune modulators andare useful in treatment or prophylaxis of pathologies mediated bylymphocyte actions including acute or chronic rejection of tissue graftssuch as organ transplants or graft vs. host disease as well asautoimmune diseases. Autoimmunue diseases that could be treated withcompounds of the invention include, but are not limited to: systemiclupus erythematosus, multiple sclerosis, rheumatoid arthritis,inflammatory bowel diseases including Crohn's disease and ulcerativecolitis, type I diabetes, uveitis, psoriasis and myasthenia gravis. Thecompounds of the invention are useful also in treating inflammatorydisorders such as atopic asthma, inflammatory glomerular injury andischemia-reperfusion injury.

Compounds of formula XII wherein R₁₅ is hydroxy are primary alcohols. Itis hypothesized that the hydroxy moiety of the primary alcohols isconverted to phosphates in vivo. Therefore, the primary alcohol S1Panalogs of the present invention are expected to serve as prodrug formsof active S1P receptor modulating compounds. Therefore, in accordancewith one embodiment pharmaceutical compositions comprising the primaryalcohol S1P analogs of the present invention are administered to treatpatients for a variety of ailments or conditions, including the use ofthe compounds for immuno-modulation to prevent or diminish tissue graftrejection.

S1P is metabolized by a variety of conceivable routes includingphosphatases, esterases or transported into cells. The S1P signal atreceptors might be prolonged if the routes of degradation could beevaded or inhibited by S1P structural analogs. The S1P analogs of thepresent invention can be used, in accordance with one embodiment, toinhibit or evade endogenous S1P metabolic pathways includingphosphotases, esterases, transporters and S1P acyl transferases. Forexample, those S1P analogs that lack an ester bond would be resistant todegradation by endogenous esterases. One embodiment of the presentinvention is directed to compounds that function as a S1P receptoragonists and antagonists that are resistant to hydrolysis by lipidphosphate phosphatases (LPPs) or are sub-type selective inhibitors ofLPPs, and in particular are resistant to hydrolysis by sphingosine1-phosphate phosphohydrolase. Previously described S1P mimetics containa phosphate group, and thus are likely susceptible to hydrolysis byLPPs.

Alpha hydroxy phosphonates are well known phosphate mimetics. Forexample, the compounds used clinically to treat osteoporosis(pamidronate, alendronate) are alpha hydroxy bisphosphonates that areanalogs of pyrophosphate. S1P analogs can be prepared wherein thephosphate moiety is replaced by an alpha hydroxy phosphonate.Accordingly, one aspect of the present invention is directed to lipidphosphate phosphatase resistant S1P analogs having the generalstructures of Formula IX or I wherein R₄ or R₁₅, respectively, areselected from the group consisting of

The compounds of the present invention can be used for immuno-modulationas well as in anti-angiogenesis therapy, most particularly as applied intherapy of neoplastic disease. In another embodiment, the SP1 analogs ofthe present invention are used in the protection of female gonads duringradiation therapy such as applied to the abdomen in the course oftreatment of neoplastic diseases.

Lysophospholipids, sphingosine-1-phosphate (S1P) and lysophosphatidicacid (LPA), stimulate cellular proliferation and affect numerouscellular functions by signaling through G protein-coupled endothelialdifferentiation gene-encoded (S1P) receptors. Accordingly, the S1Preceptor agonists disclosed in the present invention are anticipated tohave utility in a variety of clinical settings including but not limitedto the acceleration of wound healing (including corneal wounds), thepromotion of myelination (oligodendrocyte cell function) and forimmuno-modulation. In particular, LPA has been reported (Balazs et al.Am J Physiol Regul Integr Comp Physiol, 2001 280(2):R466-472) as havingactivity in accelerating wound closing and increasing neoepithelialthickness.

In accordance with one embodiment of the present invention, apharmaceutical composition comprising one or more of the S1P receptoragonists of the present invention is administered to a mammalian species(including humans) to enhance wound repair, improve neuronal function orenhance an immune response of that species. It has also been reportedthat S1P inhibits fibrosis in various organs. Accordingly, the S1Preceptor agonists of the present invention can be used to prevent/treatdiseases associated with fibrosis of organs such as pulmonary fibrosis,interstitial pneumonia, chronic hepatitis, hepatic cirrhosis, chronicrenal insufficiency or kidney glomerular sclerosis. In one embodiment, acomposition comprising an S1P receptor agonist of the present inventionis used to treat wounds, including burns, cuts, lacerations, surgicalincisions, bed sores, and slow-healing ulcers such as those seen indiabetics. Typically the composition is administered locally as atopical formulation, however other standard routes of administration arealso acceptable.

In addition, it is believed that the S1P analogs of the presentinvention mobilize lymphocytes and increase their homing to secondarylymphoid tissues. Thus, the present analogs can be used to directlymphocytes away from transplanted organs (allografts) or healthy cells(e.g. pancreatic islets (type I diabetes), myelin sheathing (multiplesclerosis)), or other tissues that may be subjected to an undesirableimmuno response and thus decrease damage to such tissues from the immunesystem.

In another embodiment, the S1P receptor modulating compounds of thepresent invention are administered to a subject to treat or prevent adisorder of abnormal cell growth and differentiation as well asinflammatory diseases. These disorders include, but are not limited to,Alzheimer's disease, aberrant corpus luteum formation, osteoarthritis,osteoporosis, anovulation, Parkinson's disease, multiple sclerosis,rheumatoid arthritis and cancer. In accordance with one embodiment, anS1P antagonist is administered to a patient to treat a diseaseassociated with abnormal growth. In one embodiment, a compositioncomprising a compound of the general structure:

-   -   wherein R₁₁ is C₅-C₁₈ alkyl or C₅-C₁₈ alkenyl located in the        meta or para position;    -   Q is selected from the group consisting of C₃-C₆ optionally        substituted cycloalkyl, C₃-C₆ optionally substituted        heterocyclic, C₃-C₆ optionally substituted aryl C₃-C₆ optionally        substituted heteroaryl, CH₂CH₂ and —NH(CO)—;    -   R₃ is selected from the group consisting of H, C₁-C₄ alkyl and        (C₁-C₄ alkyl)OH;    -   R₂₃ is selected from the group consisting of H, F, CO₂H, OH,        C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;    -   R₂₄ is selected from the group consisting of H, F and PO₃H₂, or        R₂₃ together with R₂₄ and the carbon to which they are attached        form a carbonyl group; and    -   R₁₅ is selected from the group consisting of hydroxy,        phosphonate, and

-   -   wherein X and R₁₂ is selected from the group consisting of O and        S;    -   or a pharmaceutically acceptable salt or tautomer thereof and    -   a pharmaceutically acceptable carrier is administered to treat a        patient suffering from a disease associated with abnormal cell        growth.

In one embodiment, the compound of Formula XI is administered to treat apatient suffering from a disease associated with abnormal cell growthwherein Q is —NH(CO)—,

-   -   R₂₄ is H;    -   R₂₃ is H or C₁-C₄ alkyl;    -   R₁₅ is selected from the group consisting of hydroxy and

-   -   wherein R₁₂ is O or S, and in a further embodiment Q is

-   -   R₁₅ is OH;    -   or a pharmaceutically acceptable salt or tautomer thereof.

In addition, it is believed that the S1P analogs of the presentinvention mobilize lymphocytes and increase their homing to secondarylymphoid tissues. Thus, the present analogs can be used to directlymphocytes away from transplanted organs (allografts) or healthy cells(e.g., pancreatic islets (type I diabetes), myelin sheathing (multiplesclerosis)), or other tissues that may be subjected to an undesirableimmuno response and thus decrease damage to such tissues from the immunesystem.

In accordance with one embodiment, the S1P analogs of the presentinvention are used for immuno-modulation, wherein immuno-modulationrefers to an affect on the functioning of the immune system and includeslymphocyte trafficking. In accordance with one embodiment, an S1P analogof the present invention that exhibits potent agonist activity at S1P1is administered to a warm blooded vertebrate, including a human, toinduce immuno-modulation in a patient in need thereof. In one embodimentthe S1P analog is specific or has enhanced activity at the S1P1 receptorsubtype relative to one or more of the other S1P receptor subtypes.

In one embodiment of the present invention, the S1P analogs of thepresent invention are used as immuno-modulators to alter immune systemactivities and prevent damage to healthy tissue that would otherwiseoccur in autoimmune diseases and in organ transplantation. Inparticular, the compounds can be administered to patients as part of thetreatment associated with organ transplantation, including pancreas,pancreatic islets, kidney, heart and lung transplantations. The S1Panalogs can be administered alone or in combo with knownimmuno-suppressants such as cyclosporine, tacrolimus, rapamycin,azathioprine, cyclophosphamide, methotrexate and corticosteroids such ascortisolo, cortisone, desoxymetasone, betametasone, desametasone,flunisolide, prednisolone, prednisone, amcinomide desonide,methylprednisolone, triamcinolone, and alclometasone.

Additionally, the S1P analogs of the present invention can beadministered to patients suffering from an autoimmune disease to treatthat disease. Examples of diseases considered to be autoimmune in natureare: type I diabetes, systemic lupus erythematosus, multiple sclerosis,rheumatoid arthritis, inflammatory bowel disease including colitis andCrohn's disease, glomerulonephritis, uveitis, Hashimoto's thyroiditis,myasthenia gravis, autoimmune hemolytic anemia, autoimmunethrombocytopenic purpura, autoimmune hepatitis and Wegner's granuloma.

In accordance with one embodiment, an immuno-modulation therapy isprovided for treating mammals, including humans, in need thereof. Themethod comprises the steps of administering to said mammal an effectiveamount of a compound represented by the formula:

-   -   wherein    -   W is CR₂₇R₂₈ or (CH₂)_(n)NH(CO);    -   wherein R₂₇ and R₂₈ are independently selected from the group        consisting of H, halo and hydroxy;    -   Y is selected from the group consisting of a bond, CR₉R₁₀,        carbonyl, NH, O or S;    -   wherein R₉ and R₁₀ are independently selected from the group        consisting of H, halo, hydroxy and amino;    -   Z is CH₂, aryl, halo substituted aryl or heteroaryl;    -   R₁₁ and R₁₆ are independently selected from the group consisting        of C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, C₂-C₁₈ alkynyl, C₅-C₁₈ alkoxy,        (CH₂)_(p)O(CH₂)_(q), C₅-C₁₀ (aryl)R₂₀, C₅-C₁₀ (heteroaryl)R₂₀,        C₅-C₁₀ (cycloalkyl)R₂₀, C₅-C₁₀ alkoxy(aryl)R₂₀, C₅-C₁₀        alkoxy(heteroaryl)R₂₀ and C₅-C₁₀ alkoxy(cycloalkyl)R₂₀;    -   wherein R₂₀ is H or C₁-C₁₀ alkyl;    -   R₂₉ is H or halo;    -   R₁₇ is selected from the group consisting of H, halo, NH₂, C₁-C₆        alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₁-C₆ alkylcyano and        C₁-C₆ alkylthio;    -   R₂ and R₂₁ are both NH₂;    -   R₃ is selected from the group consisting of H, C₁-C₆ alkyl,        (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;    -   R₂₂ is selected from the group consisting of C₁-C₆ alkyl, (C₁-C₄        alkyl)OH and (C₁-C₄ alkyl)NH₂;    -   R₂₃ is selected from the group consisting of H, F, CO₂H, OH,        C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;    -   R₂₄ is selected from the group consisting of H, F and PO₃H₂, or        R₂₃ together with R₂₄ and the carbon to which they are attached        form a carbonyl group;    -   R₂₅, R₇ and R₈ are independently selected from the group        consisting of O, S, CHR₂₆, CHR₂₆, NR₂₆, and N;    -   wherein R₂₆ is H, F or C₁-C₄ alkyl;    -   R₁₅ is selected from the group consisting of hydroxy,        phosphonate, and

-   -   wherein R₁₂ is selected from the group consisting of O, NH and        S;    -   X is selected from the group consisting of O, NH and S;    -   y and m are integers independently ranging from 0 to 4;    -   p and q are integers independently ranging from 1 to 10;    -   n is an integer ranging from 0 to 10;    -   or a pharmaceutically acceptable salt or tautomer thereof, with        the proviso that W and Y are not both methylene. In one        embodiment, the compound has the general structure of Formula        II-VII as described herein to treat a patient by suppressing the        immune system and diminishing damage to healthy tissue that        would otherwise occur in autoimmune diseases and in organ        transplantation.

In one embodiment, the immuno-modulating compound has the generalstructure:

-   -   wherein R₆ is selected from the group consisting of C₁-C₁₀ alkyl        and R₂ and R₃ are independently selected from the group        consisting of H, and NH₂ with the proviso that R₂ and R₃ are not        the same, and either R₂ or R₃ is NH₂; R₂₁ is selected from the        group consisting of C₁-C₆ alkyl, (C₁-C₄ alkyl)OH and (C₁-C₄        alkyl)NH₂; and R₁₅ is selected from the group consisting of        hydroxy, phosphonate, and

-   -   wherein R₁₂ is selected from the group consisting of O, NH and        S; as well as pharmaceutically acceptable salts or tautomers of        such compounds.

The dosage to be used is, of course, dependent on the specific disorderto be treated, as well as additional factors including the age, weight,general state of health, severity of the symptoms, frequency of thetreatment and whether additional pharmaceuticals accompany thetreatment. The dosages are in general administered several times per dayand preferably one to three times per day. The amounts of the individualactive compounds are easily determined by routine procedures known tothose of ordinary skill in the art.

S1P also acts as a survival factor in many cell types. In particular,S1P receptor agonists are anticipated to have activity in protectingcells and tissues from hypoxic conditions. In accordance with oneembodiment, the S1P antagonists of the present invention areadministered to treat cells and tissues exposed to hypoxic conditions,including injury sustained as a result of ischemia. In accordance withone embodiment, the S1P analogs exhibiting S1P receptor antagonistactivity can be used to treat ischemia reperfusion type injury.Interference with the supply of oxygenated blood to tissues is definedas ischemia. The effects of ischemia are known to be progressive, suchthat over time cellular vitality continues to deteriorate and tissuesbecome necrotic. Total persistent ischemia, with limited oxygenperfusion of tissues, results in cell death and eventually incoagulation-induced necrosis despite reperfusion with arterial blood. Asubstantial body of evidence indicates that a significant proportion ofthe injury associated with ischemia is a consequence of the eventsassociated with reperfusion of ischemic tissues, hence the termreperfusion injury.

The present invention is also directed to pharmaceutical compositionscomprising the S1P receptor modulating compounds of the presentinvention. More particularly, such S1P receptor agonists and antagonistscan be formulated as pharmaceutical compositions using standardpharmaceutically acceptable carriers, fillers, solublizing agents andstabilizers known to those skilled in the art. Pharmaceuticalcompositions comprising the S1P receptor agonists and/or antagonists areadministered to an individual in need thereof by any number of routesincluding, but not limited to, topical, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, or rectal means. The oralroute is typically employed for most conditions requiring the compoundsof the invention. Preference is given to intravenous injection orinfusion for the acute treatments. For maintenance regimens the oral orparenteral, e.g. intramuscular or subcutaneous, route is preferred. Inaccordance with one embodiment, a composition is provided that comprisesan S1P analog of the present invention and albumin, more particularly,the composition comprises an S1P analog of the present invention, apharmaceutically acceptable carrier and 0.1-1.0% albumin. Albuminfunctions as a buffer and improves the solubility of the compounds.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. In accordance with oneembodiment, a kit is provided for treating a patient in need ofimmuno-modulation. In this embodiment the kit comprises one or more ofthe S1P analogs of the present invention and may also include one ormore known immuno-supressants. These pharmaceuticals can be packaged ina variety of containers, e.g., vials, tubes, microtiter well plates,bottles, and the like. Other reagents can be included in separatecontainers and provided with the kit; e.g., positive control samples,negative control samples, buffers, cell culture media, etc. Preferably,the kits will also include instructions for use.

The present invention is also directed to methods for discoveringagonists and antagonists of the interaction between S1P and the S1Preceptor. Such compounds are identified by using an assay for detectingS1P receptor activity (such as the [Y-35 S]GTP binding assay) andassaying for activity in the presence of S1P and the test compound. Moreparticularly, in the method described by Traynor and Nahorski, 1995,Mol. Pharmacol. 47: 848-854, incorporated herein by reference, G-proteincoupling to membranes can be evaluated by measuring the binding oflabeled GTP.

For example, samples comprising membranes isolated from cells expressingan S1P polypeptide can be incubated in a buffer promoting binding of thepolypeptide to ligand (i.e. S1P), in the presence of radiolabeled GTPand unlabeled GDP (e.g., in 20 mM HEPES, pH 7.4, 100 mM NaCl, and 10 mMMgCl₂, 80 pM ³⁵S-GTPγS and 3 μM GDP), with and without a candidatemodulator. The assay mixture is incubated for a suitable period of timeto permit binding to and activation of the receptor (e.g., 60 minutes at30° C.), after which time unbound labeled GTP is removed (e.g., byfiltration onto GF/B filters). Bound, labeled GTP can be measured byliquid scintillation counting. A decrease of 10% or more in labeled GTPbinding as measured by scintillation counting in a sample containing acandidate modulator, relative to a sample without the modulator,indicates that the candidate modulator is an inhibitor of S1P receptoractivity.

A similar GTP-binding assay can be performed without the presence of theligand (i.e. S1P) to identify agents that act as agonists. In this case,ligand-stimulated GTP binding is used as a standard. An agent isconsidered an agonist if it induces at least 50% of the level of GTPbinding induced by S1P when the agent is present at 10 uM or less, andpreferably will induce a level which is the same as or higher than thatinduced by ligand.

GTPase activity can be measured by incubating cell membrane extractscontaining an S1P receptor with γ³²P-GTP. Active GTPase will release thelabel as inorganic phosphate, which can be detected by separation offree inorganic phosphate in a 5% suspension of activated charcoal in 20mM H₃PO₄, followed by scintillation counting. Controls would includeassays using membrane extracts isolated from cells not expressing an S1Preceptor (e.g., mock-transfected cells), in order to exclude possiblenon-specific effects of the candidate modulator. In order to assay forthe effect of a candidate modulator on S1P-regulated GTPase activity,cell membrane samples can be incubated with a ligand (e.g., S1P), withand without the modulator, and a GTPase assay can be performed asdescribed above. A change (increase or decrease) of 10% or more in thelevel of GTP binding or GTPase activity relative to samples withoutmodulator is indicative of S1P modulation by a candidate modulator.

Identified S1P receptor agonists and antagonists can be used to treat avariety of human diseases and disorders, including, but not limited tothe treatment of infections such as bacterial, fungal, protozoan andviral infections, particularly infections caused by HIV-1 or HIV-2;pain; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson'sdisease; acute heart failure; hypotension; hypertension; urinaryretention; osteoporosis; angina pectoris; myocardial infarction; stroke;ulcers; asthma; allergy; benign prostatic hypertrophy; migraine;vomiting; psychotic and neurological disorders, including anxiety,schizophrenia, manic depression, depression, delirium, dementia, andsevere mental retardation.

EXAMPLE 1

Chemical Syntheses of S1P Analogs

To develop good mimetics for S1P, a synthetic route was designed thathad several aspects in mind (Scheme 1). First, butoxycarbonyl protectedL-serine was chosen as starting material primarily because itretrosynthetically resembled the linker region of S1P. In addition, thestarting material is a cheap and commercially available protected aminoacid. Secondly, chemodivergence was taken into consideration. Couplingof the long chain was performed late in the synthesis so that severalchain lengths could be prepared from a common intermediate. Anotherimportant issue to address was the overwhelming insolubility of thefinal compounds. Due to this insolubility, the target molecules couldnot be purified by chromatography or crystallization methods, nor couldthey tolerate a simple workup. It was therefore necessary to design afinal step that quantitatively generated only the target product, andallowed for removal of excess reagents under vacuum. This wasaccomplished by employing trifluoroacetic acid deprotection at the endof the route.

The syntheses of the S1P analogs described in the synthetic schemes ofExample 1 were accomplished using solvents purified by filtrationthrough alumina (activity J) and unless otherwise indicated allreactions were conducted at room temperature. All reactions wereperformed under an inert atmosphere and all products were purified using230-400 mesh silica gel. Each product was analyzed by thin layerchromatography (single spot) and spectroscopic methods including ¹H NMR,¹³C NMR, and mass spectrometry. The assigned structures of the S1Panalogs were consistent with all spectral data obtained. All finalproducts were obtained as the TFA salts.

Synthesis of (2S) S1P Analogs VPC22041, 51, 53, and 63

% Yields Compound R A B C D E VPC22041 n-C₁₂H₂₅NH 100 100 91 33 100VPC22051 n-C₁₄H₂₉NH 100 100 91 41 96 VPC22053 n-C₁₄H₂₉O 100 100 91 15100 VPC22063 n-C₁₆H₃₃NH 100 100 91 26 100

Benzyl protection of N-Boc serine. To a stirring solution ofN-Boc-(L)-Serine (4.87 mmol) in DMF (100 mL) was added cesium carbonate(5.11 mmol) and stirring was continued 30 min. Benzyl bromide (5.84mmol) was then added and the resulting solution was stirred 12 h. Thereaction mixture was then diluted with ethyl acetate (25 mL), washedwith lithium bromide (3×15 mL), sodium bicarbonate (2×15 mL), and brine(2×15 mL). The organic layer was dried over sodium sulfate. The solventwas then removed under reduced pressure and the resulting tan oil waspurified by flash chromatography, using 1:1 petroleum ether/diethylether, to afford the product (100%) as a white solid. R_(f)=0.26 (1:1petroleum ether/diethyl ether).

Phosphorylation of resulting alcohol. For phosphorylation, reaction isperformed in the absence of light, work up and columns are completedwith as little light as possible. To a solution of the benzyl protectedserine (1.98 mmol) in 1:1 CH₂Cl₂/THF (50 mL) was added tetrazole (3.96mmol) and the resulting mixture was stirred 30 min.Di-tert-butyl-di-isopropylphosphoramidite (3.96 mmol) was then added andthe resulting reaction mixture was stirred 15 h. Hydrogen peroxide (7.92mmol) was then added and the resulting mixture was stirred 3 h, cooledto 0° C., and quenched by addition of aqueous Na₂S₂O₅. The resultingsolution was diluted with ethyl acetate (100 mL) and extracted with 50%aqueous Na₂S₂O₅ (2×20 mL). The organic layer was dried over sodiumsulfate and the solvent was removed under reduced pressure to afford atan oil. Flash chromatography, using 90:10 CHCl₃/acetone, provided theproduct (97%) as a clear oil. R_(f)=0.67 (90:10 CHCl₃/acetone).

Debenzylation of phosphorylated serine. To a solution of thephosphorylated serine (1.55 mmol) in 200 proof ethanol (25 mL) was addeda catalytic amount of palladium on activated carbon. To the resultingsolution was applied a positive pressure of hydrogen gas and thereaction mixture was stirred 12 h. The reaction mixture was thenfiltered through a plug of celite eluting with methanol and the solventwas removed under reduced pressure to yield the product (91%) as aslightly yellow oil. R_(f)=0 (90:10 CHCl₃/methanol).

Coupling of long chain amine with phosphorylated acid. A solution of theacid (0.252 mmol), a catalytic amount of 4-dimethylaminopyridine,1-hydroxybenzotriazole hydrate (0.277 mmol), the long chain amine oralcohol (0.252 mmol), and 15 mL of CH₂Cl₂ was cooled to 0° C. withstirring. To the resulting solution at 0° C. was addeddicyclohexylcarbodiimide (0.277 mmol) and the mixture was allowed toreturn to rt. with stirring continuing for 12 h. The reaction mixturewas then recooled to 0° C. and filtered. The filtrate washed with sodiumbicarbonate (3×10 mL), ammonium chloride (3×10 mL), and the organiclayers were dried over sodium sulfate. The solvent was then removedunder reduced pressure and the resulting yellow oil was purified byflash chromatography to afford the product.

-   -   VPC22041: 33%, white solid, R_(f)=0.78 (90:10 CHCl₃/methanol).    -   VPC22051: 41%, white solid, R_(f)=0.80 (90:10 CHCl₃/methanol).    -   VPC22053: 15%, white solid, R_(f)=0.20 (95:5 CHCl₃/acetone).    -   VPC22063: 26%, white solid, R_(f)=0.79 (90:10 CHCl₃/methanol).

Deprotection of N-Boc and phosphate groups. To a stirred solution of theprotected final product (0.072 mmol) in CH₂Cl₂ (1 mL) was addedtrifluoroacetic acid (12.98 mmol) and stirring was continued 4 h. Underreduced pressure, solvent and excess trifluoroacetic acid were removedaffording a brown oil. The oil was rinsed with ether and the solvent wasremoved under vacuum 5 times to afford the product.

-   -   VPC22041: 100%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).    -   VPC22051: 96%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).    -   VPC22053: 100%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).    -   VPC22063: 100%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).

For S1P analog VPC22051 the PyBOP coupling procedure (as used inVPC22135) was used in place of DCC coupling. The product was obtained in15% yield as a clear oil.

Synthesis of (2R) S1P Analog VPC22135

Coupling of long chain amine with protected serine. To a stirringsolution of N-Boc-(D)-Serine-OBn (0.847 mmol) in CH₂Cl₂ (20 mL) wasadded PyBOP (0.847 mmol) followed by diisopropylethylamine (0.847 mmol).After 5 min. of stirring, 1-tetradecylamine (0.847 mmol) was added andstirring was continued for 1 h after which time more 1-tetradecylaminewas added (0.254 mmol). Stirring was continued for another 3 h and thenthe reaction mixture was diluted with ethyl acetate (20 mL) and washedwith sodium bicarbonate (3×15 mL), ammonium chloride (2×15 mL), and theorganic layer was dried over sodium sulfate. Solvents were removed underreduced pressure to afford a clear gelatinous solid, which was purifiedby flash chromatography, using 95:5 CHCl₃/methanol, to afford theproduct (68%) as a white solid. R_(f)=0.78 (95:5 CHCl₃/methanol).

Benzyl deprotection of coupled product. To a solution of the coupledproduct (0.579 mmol) in 200 proof ethanol (15 mL) was added a catalyticamount of palladium on activated carbon. To the resulting solution wasapplied a positive pressure of hydrogen gas and the reaction mixture wasstirred 12 h. The reaction mixture was then filtered through a plug ofcelite eluting with methanol and then the solvent was removed underreduced pressure to yield the product (87%) as a clear oil. R_(f)=0.5(95:5 CHCl₃/methanol).

Phosphorylation of resulting alcohol. For phosphorylation, reaction isperformed in the absence of light, work up and columns are completedwith as little light as possible. To a solution of the alcohol (0.474mmol) in 1:1 CH₂Cl₂/THF (20 mL) was added tetrazole (0.948 mmol) and theresulting mixture was stirred 30 min.Di-tert-butyl-di-isopropylphosphoramidite (0.948 mmol) was then addedand the resulting reaction mixture was stirred 15 h. Hydrogen peroxide(1.896 mmol) was then added and the resulting mixture was then stirred24 h, cooled to 0° C., and quenched by addition of aqueous Na₂S₂O₅. Theresulting solution was diluted with ethyl acetate (50 mL) and washedwith sodium bicarbonate (2×15 mL), water (1×15 mL), and finally brine(1×15 mL). The organic layer was dried over sodium sulfate and thesolvent was removed under reduced pressure to afford a clear oil. Flashchromatography, using 90:10 CHCl₃/acetone, provided the product (100%)as a clear oil. R_(f)=0.23 (90:10 CHCl₃/acetone).

Deprotection of N-Boc and phosphate groups. To a stirred solution of theprotected product (0.071 mmol) in CH₂Cl₂ (1 mL) was addedtrifluoroacetic acid (12.98 mmol) and stirring was continued 4 h. Underreduced pressure, solvent and excess trifluoroacetic acid were removedaffording a brown oil. Rinsed oil with ether and removed under vacuum 5times to afford the product (56%) as a white solid. R_(f)=0 (90:10CHCl₃/methanol).

Synthesis of (2R) S1P Analog VPC22157, 173, 199, and 211

Coupling of long chain aniline with protected serine. To a stirringsolution of N-Boc-(D)-Serine-OBn (0.339 mmol) in CH₂Cl₂ (10 mL) wasadded PyBOP (0.339 mmol) followed by diisopropylethylamine (0.339 mmol).After 5 min. of stirring, the aniline (0.339 mmol) was added andstirring was continued for 4 h. The reaction mixture was then dilutedwith ethyl acetate (10 mL) and washed with sodium bicarbonate (3×10 mL),ammonium chloride (2×10 mL), and the organic layer was dried over sodiumsulfate. Solvents were removed under reduced pressure to afford a cleargelatinous solid, which was purified by flash chromatography to affordthe product.

-   -   VPC22157: 77%, white solid, R_(f)=0.80 (90:10 CHCl₃/acetone).    -   VPC22173: 73%, white solid, R_(f)=0.78 (90:10 CHCl₃/acetone).    -   VPC22199: 65%, white solid, R_(f)=0.79 (90:10 CHCl₃/acetone).    -   VPC22211: 71%, white solid, R_(f)=0.80 (90:10 CHCl₃/acetone).

Benzyl deprotection of coupled product. To a solution of the coupledproduct (0.260 mmol) in 200 proof ethanol (10 mL) was added a catalyticamount of palladium on activated carbon. To the resulting solution wasapplied a positive pressure of hydrogen gas and the reaction mixture wasstirred 12 h. The reaction mixture was then filtered through a plug ofcelite eluting with methanol and then the solvent was removed underreduced pressure to yield the product.

-   -   VPC22157: 85%, clear oil, R_(f)=0.50 (95:5 CHCl₃/methanol).    -   VPC22173: 60%, clear oil, R_(f)=0.55 (95:5 CHCl₃/methanol).    -   VPC22199: 70%, clear oil, R_(f)=0.48 (95:5 CHCl₃/methanol).    -   VPC22211: 9%, clear oil, R_(f)=0.53 (95:5 CHCl₃/methanol).

Phosphorylation of resulting alcohol. For phosphorylation, reaction isperformed in the absence of light, work up and columns are completedwith as little light as possible. To a solution of the alcohol (0.220mmol) in 1:1 CH₂Cl₂/THF (10 mL) was added tetrazole (0.400 mmol) and theresulting mixture was stirred 30 min.Di-tert-butyl-di-isopropylphosphoramidite (0.400 mmol) was then addedand the resulting reaction mixture was stirred 15 h. Hydrogen peroxide(0.800 mmol) was then added and the resulting mixture was then stirred24 h, cooled to 0° C., and quenched by addition of aqueous Na₂S₂O₅. Theresulting solution was diluted with ethyl acetate (25 mL) and washedwith sodium bicarbonate (2×10 mL), water (1×10 mL), and finally brine(1×10 mL). The organic layer was dried over sodium sulfate and thesolvent was removed under reduced pressure to afford a clear oil. Flashchromatography provided the product as a clear oil.

-   -   VPC22157: 84%, clear oil, R_(f)=0.23 (90:10 CHCl₃/acetone).    -   VPC22173: 96%, clear oil, R_(f)=0.30 (90:10 CHCl₃/acetone).    -   VPC22199: 87%, clear oil, R_(f)=0.72 (80:20 CHCl₃/acetone).    -   VPC22211: 90%, clear oil, R_(f)=0.58 (80:20 CHCl₃/acetone).

Deprotection of N-Boc and phosphate groups. To a stirred solution of theprotected product (0.162 mmol) in CH₂Cl₂ (2 mL) was addedtrifluoroacetic acid (25.96 mmol) and stirring was continued 4 h. Underreduced pressure, solvent and excess trifluoroacetic acid were removedaffording a brown oil. Rinsed oil with ether and removed under vacuum 5times to afford the product.

-   -   VPC22157: 100%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).    -   VPC22173: 58%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).    -   VPC22199: 75%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).    -   VPC22211: 100%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).        Synthesis of (2S) S1P Analogs VPC22179 and 181

Benzyl protection of N-Boc serine. To a stirring solution ofN-Boc-(L)-Serine (2.44 mmol) in DMF (50 mL) was added cesium carbonate(2.56 mmol) and stirring was continued 30 min. Benzyl bromide (2.92mmol) was then added and the resulting solution was stirred 12 h. Thereaction mixture was then diluted with ethyl acetate (15 mL), washedwith lithium bromide (3×10 mL), sodium bicarbonate (2×10 mL), and brine(2×10 mL). The organic layer was dried over sodium sulfate. The solventwas then removed under reduced pressure and the resulting tan oil waspurified by flash chromatography, using 1:1 petroleum ether/diethylether, to afford the product (100%) as a white solid. R_(f)=0.26 (1:1petroleum ether/diethyl ether).

Phosphorylation of resulting alcohol. For phosphorylation, reaction isperformed in the absence of light, work up and columns are completedwith as little light as possible. To a solution of the benzyl protectedserine (2.22 mmol) in 1:1 CH₂Cl₂/THF (100 mL) was added tetrazole (4.43mmol) and the resulting mixture was stirred 30 min.Di-tert-butyl-di-isopropylphosphoramidite (4.43 mmol) was then added andthe resulting reaction mixture was stirred 15 h. Hydrogen peroxide (8.86mmol) was then added and the resulting mixture was stirred 3 h, cooledto 01 C, and quenched by addition of aqueous Na₂S₂O₅. The resultingsolution was diluted with ethyl acetate (100 mL) and extracted with 50%aqueous Na₂S₂O₅ (2×20 mL). The organic layer was dried over sodiumsulfate and the solvent was removed under reduced pressure to afford atan oil. Flash chromatography, using 90:10 CHCl₃/acetone, provided theproduct (97%) as a clear oil. R_(f)=0.67 (90:10 CHCl₃/acetone).

Debenzylation of phosphorylated serine. To a solution of thephosphorylated serine (1.55 mmol) in 200 proof ethanol (25 mL) was addeda catalytic amount of palladium on activated carbon. To the resultingsolution was applied a positive pressure of hydrogen gas and thereaction mixture was stirred 12 h. The reaction mixture was thenfiltered through a plug of celite eluting with methanol and the solventwas removed under reduced pressure to yield the product (91%) as aslightly yellow oil. R_(f)=0 (90:10 CHCl₃/methanol).

Coupling of long chain aniline with phosphorylated acid. To a stirringsolution of the phosphorylated acid (0.252 mmol) in CH₂Cl₂ (10 mL) wasadded PyBOP (0.252 mmol) followed by diisopropylethylamine (0.252 mmol).After 5 min. of stirring, the aniline (0.252 mmol) was added andstirring was continued for 4 h. The reaction mixture was then dilutedwith ethyl acetate (10 mL) and washed with sodium bicarbonate (3×10 mL),ammonium chloride (2×10 mL), and the organic layer was dried over sodiumsulfate. Solvents were removed under reduced pressure to afford theproduct.

-   -   VPC22179: 43%, white solid, R_(f)=0.40 (90:10 CHCl₃/acetone).    -   VPC22181: 60%, white solid, R_(f)=0.35 (90:10 CHCl₃/acetone).

Deprotection of N-Boc and phosphate groups. To a stirred solution of theprotected final product (0.117 mmol) in CH₂Cl₂ (1.5 mL) was addedtrifluoroacetic acid (19.48 mmol) and stirring was continued 4 h. Underreduced pressure, solvent and excess trifluoroacetic acid were removedaffording a brown oil. The oil was rinsed with ether and the solvent wasremoved under vacuum 5 times to afford the product.

-   -   VPC22179: 100%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).    -   VPC22181: 100%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).        Synthesis of (2R) S1P Analog VPC22277

Tosyl protection of the long chain aniline. To a stirring solution ofthe 4-decylaniline (0.428 mmol) in pyridine (3 mL) under inertatmosphere at 0° C. was added tosyl chloride (0.428 mmol). The reactionmixture was warmed to r.t. After 20 min., the reaction mixture wasdiluted with water (10 mL) and ethyl acetate (10 mL). The aqueous layerwas discarded and the organic layer washed with 1N HCl (3×10 mL), sat.sodium bicarbonate (3×10 mL) and brine (2×10 mL). The organic layer wasdried over sodium sulfate and the solvent was removed under reducedpressure to yield the product (81%) as pink crystals, which needed nofurther purification. R_(f)=0.82 (90:10 CHCl₃/acetone).

Reduction of protected amino acid. At −10° C., under inert atmosphere,N-Boc-(D)-Ser-OBz (0.678 mmol) and diisopropylethylamine (0.678 mmol)were added to stirring THF (3 mL). Isobutylchloroformate (0.745 mmol)was then slowly added. The reaction mixture was allowed to stir for 1 huntil a precipitate was observed. The reaction mixture was then filteredand the filtrate was re-cooled to −10° C. Meanwhile, sodium borohydride(1.36 mmol) was dissolved in stirring water (0.5 mL) under inertatmosphere and this mixture was cooled to −10° C. The original reactionmixture was then cannulated into the sodium borohydride mixture slowlyand the newly formed reaction mixture was brought to r.t. and stirred 1h. The reaction mixture was then quenched by addition of sat. ammoniumchloride (5 mL), diluted with ethyl acetate (15 mL) and the aqueouslayer was discarded. The organic layer was then washed with sat.ammonium chloride (3×10 mL), sat. sodium bicarbonate (3×10 mL) andfinally brine (1×10 mL). The organic layer was dried over sodium sulfateand the solvent was removed under reduced pressure to yield the crudeproduct as a white solid. The crude product was purified by flashchromatography, using 80:20 CHCl₃/acetone, to afford the product (42%)as a white solid. R_(f)=0.48 (80:20 CHCl₃/acetone).

Coupling of aniline with alcohol. To a stirring solution of the aniline(0.209 mmol) in THF (3 mL) under an inert atmosphere was addedtriphenylphospine (0.254 mmol), the alcohol (0.105 mmol), and finallyDEAD (0.209 mmol). The reaction mixture was stirred 12 h and thenconcentrated to a clear oil. Petroleum ether was added to the clear oiland solid triphenylphosphine oxide was allowed to settle on the bottomof the flask. The clear petroleum ether layer was then pipetted off andconcentrated to a clear oil. The crude product was then subjected toflash chromatography, using 1:1 petroleum ether/ether, to afford thefinal product (50%) as a white solid. R_(f)=0.83 (1:1 petroleumether/ether).

Tosyl deprotection of the coupled product. Ammonia (20 mL) was condensedin a 2-neck round bottom flask equipped with a stirbar and cold fingerthat was cooled to −70° C. under an inert atmosphere. Sodium metal (4.27mmol) was then added to the reaction mixture followed by the tosylprotected amine (0.427 mmol) in THF (8 mL). The dark blue reactionmixture was stirred for 1 h at −70° C. and was then quenched withethanol until the solution was clear/white and the reaction mixture wasthen stirred at r.t. overnight. The reaction mixture was then dilutedwith ethyl acetate (20 mL) and washed with sat. ammonium chloride (3×20mL), sat. sodium bicarbonate (3×20 mL), and finally brine (1×20 mL). Theorganic layer was dried over sodium sulfate and the solvent was removedunder reduced pressure to yield the crude product as a clear oil. Thecrude product was purified by flash chromatography, using 1:1 ethylacetate/hexanes, to afford the product (40%) as a white solid.R_(f)=0.42 (1:1 ethyl acetate/hexanes).

Phosphorylation of resulting alcohol. For phosphorylation, reaction isperformed in the absence of light, work up and columns are completedwith as little light as possible. To a solution of the alcohol (0.130mmol) in 1:1 CH₂Cl₂/THF (5 mL) was added tetrazole (0.130 mmol) and theresulting mixture was stirred 30 min.Di-tert-butyl-di-isopropylphosphoramidite (0.130 mmol) was then addedand the resulting reaction mixture was stirred 15 h. Hydrogen peroxide(30%, 0.044 mL) was then added and the resulting mixture was thenstirred 24 h, cooled to 0° C., and quenched by addition of aqueousNa₂S₂O₅. The resulting solution was diluted with ethyl acetate (10 mL)and washed with sodium bicarbonate (2×10 mL), water (1×10 mL), andfinally brine (1×10 mL). The organic layer was dried over sodium sulfateand the solvent was removed under reduced pressure to afford a clearoil. Flash chromatography, using 1:1 ethyl acetate/hexanes, provided theproduct (12%) as a clear oil. R_(f)=0.41 (1:1 ethyl acetate/hexanes).

Deprotection of N-Boc and phosphate groups. To a stirred solution of theprotected final product (0.016 mmol) in CH₂Cl₂ (0.5 mL) was addedtrifluoroacetic acid (6.49 mmol) and stirring was continued 4 h. Underreduced pressure, solvent and excess trifluoroacetic acid was removedaffording a brown oil. The oil was rinsed with ether and the solvent wasremoved under vacuum 5 times to afford the product (100%) as a whitesolid. R_(f)=0 (90:10 CHCl₃/methanol).

Synthesis of (2R) S1P Analog VPC23031, 19, 65, 69, 75 and 79

% Yields Compound(s) n A B C D E F G VPC23031 4 24 66 52 100 X 90 100VPC23019 6 100 85 90 95 X 56 92 VPC23065, 69 8 34 84 84 89 100 89 86VPC23075, 79 7 66 100 100 27 93 77 100

Coupling of aryl halide with terminal alkyne. All starting materialswere thoroughly flushed with nitrogen before the reaction. To a stirringsolution of the aryl halide (2.01 mmol), bis(dibenzylideneacetone)palladium (0.04 mmol), triphenylphosphine (0.10 mmol), and copper iodide(0.04 mmol) in THF (10 mL) under inert atmosphere was added the terminalalkyne (2.21 mmol) followed by diisopropylethylamine (8.04 mmol). Thereaction mixture was then stirred at r.t. for 12 h. The reaction mixturewas then diluted with ethyl acetate (15 mL) and washed with sodiumbicarbonate (3×15 mL), ammonium chloride (3×15 mL) and finally brine(1×15 mL). The organic layer was then dried over sodium sulfate.Solvents were removed under reduced pressure to afford a tan oil. Flashchromatography provided the final product.

-   -   VPC23031: 24%, yellow oil, R_(f)=0.61 (90:10 hexanes/ether).    -   VPC23019: 100%, yellow oil, R_(f)=0.55 (90:10 hexanes/ether).    -   VPC23065, 69: 66%, yellow oil, R_(f)=0.75 (90:10 hexanes/ether).    -   VPC23075, 79: 34%, yellow oil, R_(f)=0.75 (90:10 hexanes/ether).

Reduction of the coupled product. To a solution of the coupled product(1.68 mmol) in 200 proof ethanol (10 mL) was added a catalytic amount ofpalladium on activated carbon. To the resulting solution was applied apositive pressure of hydrogen gas and the reaction mixture was stirred12 h. The reaction mixture was then filtered through a plug of celiteeluting with methanol and then the solvent was removed under reducedpressure to yield the crude product.

-   -   VPC23031: 66%, yellow solid, R_(f)=0.53 (95:5 CHCl₃/acetone).    -   VPC23019: 85%, yellow solid, R_(f)=0.55 (95:5 CHCl₃/acetone).    -   VPC23065, 69: 84%, yellow solid, R_(f)=0.79 (95:5        CHCl₃/acetone).    -   VPC23075, 79: 100%, yellow solid, R_(f)=0.80 (95:5        CHCl₃/acetone).

Coupling of long chain aniline with protected serine. To a stirringsolution of N-Boc-(D)-Serine-OBn (0.740 mmol) in CH₂Cl₂ (20 mL) wasadded PyBOP (0.740 mmol) followed by diisopropylethylamine (0.740 mmol).After 5 min. of stirring, the aniline (0.740 mmol) was added andstirring was continued for 4 hours. The reaction mixture was thendiluted with ethyl acetate (20 mL) and washed with 1 N HCl (3×20 mL),sodium bicarbonate (3×20 mL), and finally brine (1×20 mL), and theorganic layer was dried over sodium sulfate. Solvents were removed underreduced pressure to afford a clear oil, which was purified by flashchromatography to afford the product.

-   -   VPC23031: 52%, clear oil, R_(f)=0.35 (dichloromethane).    -   VPC23019: 90%, clear oil, R_(f)=0.61 (70:30 hexanes/ethyl        acetate).    -   VPC23065, 69: 84%, clear oil, R_(f)=0.82 (90:10 CHCl₃/acetone).    -   VPC23075, 79: 100%, clear oil, R_(f)=0.92 (90:10 CHCl₃/acetone).

Benzyl deprotection of coupled product. To a solution of the coupledproduct (0.667 mmol) in 200 proof ethanol (15 mL) was added a catalyticamount of palladium on activated carbon. To the resulting solution wasapplied a positive pressure of hydrogen gas and the reaction mixture wasstirred 12 h. The reaction mixture was then filtered through a plug ofcelite eluting with methanol and then the solvent was removed underreduced pressure to yield the product.

-   -   VPC23031: 100%, clear oil, R_(f)=0.27 (70:30 hexanes/ethyl        acetate).    -   VPC23019: 95%, clear oil, R_(f)=0.28 (70:30 hexanes/ethyl        acetate).    -   VPC23065, 69: 89%, clear oil, R_(f)=0.62 (1:1 hexanes/ethyl        acetate).    -   VPC23075, 79: 27%, clear oil, R_(f)=0.43 (1:1 hexanes/ethyl        acetate).

Deprotection to afford free alcohol. To a stirred solution of the N-Bocprotected alcohol (0.143 mmol) in CH₂Cl₂ (2 mL) was addedtrifluoroacetic acid (25.96 mmol) and stirring was continued 4 h. Underreduced pressure, solvent and excess trifluoroacetic acid were removedaffording a brown oil. The oil was rinsed with ether and the solvent wasremoved under vacuum 5 times to afford the product.

-   -   VPC23065: 100%, white solid, R_(f)=0.2 (90:10 CHCl₃/methanol).    -   VPC23075: 93%, white solid, R_(f)=0.2 (90:10 CHCl₃/methanol).

Phosphorylation of N-Boc protected alcohol. For phosphorylation, thereaction is performed in the absence of light, work up and columns arecompleted with as little light as possible. To a solution of the alcohol(0.247 mmol) in 1:1 CH₂Cl₂/THF (15 mL) was added tetrazole (0.495 mmol)and the resulting mixture was stirred 30 min.Di-tert-butyl-di-isopropylphosphoramidite (0.495 mmol) was then addedand the resulting reaction mixture was stirred 15 h. Hydrogen peroxide(0.989 mmol) was then added and the resulting mixture was then stirred24 h, cooled to 0° C., and quenched by addition of aqueous Na₂S₂O₅. Theresulting solution was diluted with ethyl acetate (25 mL) and washedwith sodium bicarbonate (3×15 mL), ammonium chloride (3×15 mL), andfinally brine (1×15 mL). The organic layer was dried over sodium sulfateand the solvent was removed under reduced pressure to afford a clearoil. Flash chromatography provided the product.

-   -   VPC23031: 90%, clear oil, R_(f)=0.80 (80:20 ether/ethyl        acetate).    -   VPC23019: 56%, clear oil, R_(f)=0.82 (80:20 ether/ethyl        acetate).    -   VPC23069: 89%, clear oil, R_(f)=0.85 (90:10 ether/ethyl        acetate).    -   VPC23079: 77%, clear oil, R_(f)=0.85 (90:10 ether/ethyl        acetate).

Deprotection of N-boc and phosphate groups. To a stirred solution of theprotected product (0.162 mmol) in CH₂Cl₂ (2 mL) was addedtrifluoroacetic acid (25.96 mmol) and stirring was continued 4 h. Underreduced pressure, solvent and excess trifluoroacetic acid were removedaffording a brown oil. Rinsed oil with ether and removed under vacuum 5times to afford the product.

-   -   VPC23031: 100%, clear oil, R_(f)=0 (90:10 CHCl₃/methanol).    -   VPC23019: 92%, clear oil, R_(f)=0 (90:10 CHCl₃/methanol).    -   VPC23069: 86%, clear oil, R_(f)=0 (90:10 CHCl₃/methanol).    -   VPC23079: 100%, clear oil, R_(f)=0 (90:10 CHCl₃/methanol).        Synthesis of (2R)S1P Analog VPC23087 and 89:

Coupling of aryl halide with terminal alkyne. All starting materialswere thoroughly flushed with nitrogen before the reaction. To a stirringsolution of the aryl halide (2.01 mmol), bis(dibenzylideneacetone)palladium (0.04 mmol), triphenylphosphine (0.10 mmol), and copper iodide(0.04 mmol) in THF (10 mL) under inert atmosphere was added the terminalalkyne (2.21 mmol) followed by diisopropylethylamine (8.04 mmol). Thereaction mixture was then stirred at r.t. for 12 h. The reaction mixturewas then diluted with ethyl acetate (15 mL) and washed with sodiumbicarbonate (3×15 mL), ammonium chloride (3×15 mL) and finally brine(1×15 mL). The organic layer was then dried over sodium sulfate.Solvents were removed under reduced pressure to afford a tan oil. Flashchromatography, using 70:30 hexanes/ethyl acetate provided the finalproduct (44%) as a yellow solid. R_(f)=0.79 (70:30 hexanes/ethylacetate).

Coupling of long chain aniline with protected serine. To a stirringsolution of N-boc-(D)-Serine-OBn (0.288 mmol) in CH₂Cl₂ (10 mL) wasadded PyBOP (0.288 mmol) followed by diisopropylethylamine (0.288 mmol).After 5 min. of stirring, the aniline (0.288 mmol) was added andstirring was continued for 4 hours. The reaction mixture was thendiluted with ethyl acetate (10 mL) and washed with 1 N HCl (3×10 mL),sodium bicarbonate (3×10 mL), and finally brine (1×10 mL), and theorganic layer was dried over sodium sulfate. Solvents were removed underreduced pressure to afford a clear oil. Flash chromatography, using70:30 hexanes/ethyl acetate provided the final product (65%) as a clearoil. R_(f)=0.64 (70:30 hexanes/ethyl acetate).

Benzyl deprotection and reduction of coupled product. To a solution ofthe coupled product (0.188 mmol) in 200 proof ethanol (10 mL) was addeda catalytic amount of palladium on activated carbon. To the resultingsolution was applied a positive pressure of hydrogen gas and thereaction mixture was stirred 12 h. The reaction mixture was thenfiltered through a plug of celite eluting with methanol and then thesolvent was removed under reduced pressure to yield the crude product asa clear oil. Flash chromatography, using 1:1 hexanes/ethyl acetateprovided the final product (49%) as a clear oil. R_(f)=0.51 (1:1hexanes/ethyl acetate).

Deprotection to afford free alcohol. To a stirred solution of the N-Bocprotected alcohol (0.025 mmol) in CH₂Cl₂ (1 mL) was addedtrifluoroacetic acid (12.98 mmol) and stirring was continued 4 h. Underreduced pressure, solvent and excess trifluoroacetic acid were removedaffording a brown oil. The oil was rinsed with ether and the solvent wasremoved under vacuum 5 times to afford the product (100%) as a whitesolid. R_(f)=0.2 (90:10 CHCl₃/methanol).

Phosphorylation of N-Boc protected alcohol. For phosphorylation,reaction is performed in the absence of light, work up and columns arecompleted with as little light as possible. To a solution of the alcohol(0.092 mmol) in 1:1 CH₂Cl₂/THF (10 mL) was added tetrazole (0.183 mmol)and the resulting mixture was stirred 30 min.Di-tert-butyl-di-isopropylphosphoramidite (0.183 mmol) was then addedand the resulting reaction mixture was stirred 15 h. Hydrogen peroxide(0.367 mmol) was then added and the resulting mixture was then stirred24 h, cooled to 0° C., and quenched by addition of aqueous Na₂S₂O₅. Theresulting solution was diluted with ethyl acetate (15 mL) and washedwith sodium bicarbonate (3×15 mL), ammonium chloride (3×15 mL), andfinally brine (1×15 mL). The organic layer was dried over sodium sulfateand the solvent was removed under reduced pressure to afford a clearoil. Flash chromatography, using 90:10 ethyl acetate/ether provided thefinal product (93%) as a clear oil. R_(f)=0.85 (90:10 ethylacetate/ether).

Deprotection of N-Boc and phosphate groups. To a stirred solution of theprotected product (0.063 mmol) in CH₂Cl₂ (2 mL) was addedtrifluoroacetic acid (25.96 mmol) and stirring was continued 4 h. Underreduced pressure, solvent and excess trifluoroacetic acid were removedaffording a brown oil. The oil was rinsed with ether and the solvent wasremoved under vacuum 5 times to afford the product (100%) as a whitesolid. R_(f)=0 (90:10 CHCl₃/methanol).

Synthesis of (2R) Benzimidazole Compound:

Acetylation of the aniline. To a stirring solution of acetic anhydride(10 mL) under inert atmosphere was added octyl aniline (0.738 mmol) andstirring was continued for 1 h. Sat. aqueous sodium bicarbonate was thenadded to neutralize and acetic acid present. The aqueous solution wasthen extracted with ethyl acetate (3×15 mL) and the combined organicextracts were dried over sodium sulfate and concentrated to afford thefinal product (100%) as a yellow solid that was used without furtherpurification. R_(f)=0.48 (90:10 CHCl₃/acetone).

Nitration of the acetylated aniline. To a stirring solution of aceticacid (1.08 mL), acetic anhydride (0.73 mL), and nitric acid (0.20 mL) at−15° C. under an inert atmosphere was added the acetylated aniline (0.91mmol) in approx. 1 mL of acetic acid over a period of 3 h. Reactionmixture was periodically warmed to 0° C. to avoid freezing. The reactionmixture was stirred for an additional hour and was then diluted withethyl acetate (10 mL) and neutralized using 1M NaOH and sat. aqueoussodium bicarbonate. The organic layer was removed and the aqueousportion washed twice more with ethyl acetate (10 mL each). The organiclayers were combined and dried over sodium sulfate and then concentratedto a yellow solid. Flash chromatography, using 95:5 CHCl₃/acetoneprovided the final product (100%) as a yellow solid. R_(f)=0.68 (95:5CHCl₃/acetone).

Deacetylation of the aniline. To a stirring solution of the nitrated,acetylated aniline (0.62 mmol) in ethanol (2.5 mL) under an inertatmosphere was added 40% KOH (0.13 mL). The reaction mixture was thenheated to reflux for 1 h. The solution was then cooled in ice andbrought to pH=6 using conc. HCl. This mixture was then concentrated toan orange solid and redissolved in ether (10 mL) and washed with sat.aqueous sodium bicarbonate (2×10 mL) and brine (1×10 mL). The organiclayer was then dried over sodium sulfate and concentrated to afford thefinal product (84%) as an orange solid that was used without furtherpurification. R_(f)=0.82 (95:5 CHCl₃/acetone).

Reduction of the nitro group. To a stirring solution of the nitratedaniline (0.248 mmol) in acetic acid (5 mL) was added a catalytic amountof zinc dust and stirring was continued overnight under an inertatmosphere. The reaction mixture was then diluted with ether andfiltered through a plug of celite under and inert atmosphere using etherto elute. Care was taken not to expose the ether solution to air. Thesolution was then concentrated to afford the final product (92%) as areddish-brown oil which was used directly in the next step withoutfurther purification. R_(f)=0.05 (95:5 CHCl₃/acetone).

Coupling of the diamine with protected serine. A solution ofN-boc-(D)-Serine-OBn (0.999 mmol), PyBOP (0.999 mmol),diisopropylethylamine (0.999 mmol) in CH₂Cl₂ (25 mL) was stirred 5 min.under an inert atmosphere and then cannulated into a flask containingthe diamine (0.999 mmol). This reaction mixture was then stirred 12 h.The reaction mixture was then diluted with ethyl acetate (30 mL) andwashed with sat. aqueous sodium bicarbonate (3×3 mL), ammonium chloride(3×30 mL), and finally brine (1×30 mL), and the organic layer was driedover sodium sulfate. Solvents were removed under reduced pressure toafford a brown oil. Flash chromatography, using 90:10 CHCl₃/acetoneprovided the final product (17%) as a brown oil. R_(f)=0.52 (90:10CHCl₃/acetone).

Benzyl deprotection of coupled product. To a solution of the coupledproduct (0.167 mmol) in 200 proof ethanol (10 mL) and a catalytic amountof formic acid was added a catalytic amount of palladium on activatedcarbon. To the resulting solution was applied a positive pressure ofhydrogen gas and the reaction mixture was stirred 12 h. The reactionmixture was then filtered through a plug of celite eluting with methanoland then the solvent was removed under reduced pressure to yield thecrude product as a tan oil. Prep. plate thin layer chromatography, using90:10 CHCl₃/acetone provided the final product (57%) as a tan/whitesolid. R_(f)=0.08 (90:10 CHCl₃/acetone).

Deprotection to afford free alcohol. To a stirring solution of the N-Bocprotected alcohol (0.008 mmol) in CH₂Cl₂ (0.5 mL) was addedtrifluoroacetic acid (0.5 mL) and stirring was continued 4 h. Underreduced pressure, solvent and excess trifluoroacetic acid were removedaffording a brown oil. The oil was rinsed with ether and the solvent wasremoved under vacuum 5 times to afford the product (100%) as a tansolid. R_(f)=0.2 (90:10 CHCl₃/methanol).

Phosphorylation of N-Boc protected alcohol. For phosphorylation,reaction is performed in the absence of light, work up and columns arecompleted with as little light as possible. To a solution of the alcohol(0.085 mmol) in 1:1 CH₂Cl₂/THF (5 mL) was added tetrazole (0.170 mmol)and the resulting mixture was stirred 30 min.Di-tert-butyl-di-isopropylphosphoramidite (0.170 mmol) was then addedand the resulting reaction mixture was stirred 15 h. Hydrogen peroxide(0.340 mmol) was then added and the resulting mixture was then stirred 4h, cooled to 0° C., and quenched by addition of aqueous Na₂S₂O₅. Theresulting solution was diluted with ethyl acetate (10 mL) and washedwith sodium bicarbonate (3×10 mL), ammonium chloride (3×10 mL), andfinally brine (1×10 mL). The organic layer was dried over sodium sulfateand the solvent was removed under reduced pressure to afford theproduct.

Deprotection of N-Boc and phosphate groups. To a stirring solution ofthe protected product in CH₂Cl₂ was added trifluoroacetic acid andstirring was continued 4 h. Under reduced pressure, solvent and excesstrifluoroacetic acid were removed affording a brown oil. The oil wasrinsed with ether and the solvent was removed under vacuum 5 times toafford the product.

EXAMPLE 2

All reactions for the synthetic schemes of Example 2 were accomplishedusing solvents purified by filtration through alumina (activity I)immediately prior to use. All reactions were performed under an inertatmosphere of nitrogen unless otherwise noted. All reagents werepurchased from either Aldrich (Milwaukee, Wis.), Sigma (St. Louis, Mo.),Acros (Pittsburgh, Pa.), Advanced ChemTech (Louisville, Ky.), orNovabiochem (La Jolla, Calif.). Merck silica gel F-254 precoated,aluminum backed plates were used for thin layer chromatography (TLC)analysis. Analtech Silica Gel GF 500 or 1000 μm precoated, glass backedplates were used for preparative TLC. Silicycle Ultra Pure Silica Gel(230-400 mesh) or Fisher Scientific Silica Gel 60 Sorbent (230-400 mesh)was used for column chromatography. Each product was analyzed by TLC(single spot) and spectroscopic methods including ¹H NMR, ¹³C NMR, andmass spectrometry. The nuclear magnetic resonance spectra were collectedusing a General Electric QE300 spectrometer at 300 MHz and chemicalshifts are reported in ppm. The assigned structures of the S1P analogswere consistent with all spectral data obtained.

Synthesis of Imidiazole Analog

Reagents and Conditions: (i) NaH, THF 0° to R.T. 45 min., thenSelectfluor 0° to R.T., overnight, 53%; (ii) SOCl₂, MeOH, R.T., 4-6 h.;(iii) Boc₂O, TEA, CH₂Cl₂, R.T., 4 h.; (iv) 2,2-dimethoxypropane,p-toluenesulfonic acid, CH₂Cl₂, R.T., 2 h., 62% (3 steps) (v) LiCl,NaBH₄, EtOH/THF (3:2), 0° to R.T., 4 h, 89%; (vi) PCC, CH₂Cl₂, R.T., 6h.; (vii) DBU, LiCl, CH₃CN, R.T., overnight, 40% (2 steps); (viii) Dowex50×8, EtOH, R.T. 24 h., 80%; (ix) PCC, CH₂Cl₂, R.T., 6 h. (x) NaClO₂,NaH₂PO₄.H₂O, t-butanol, 2-methyl-2-butene; (xi) p-octyl aniline, PyBOP,DIEA, CH₂Cl₂, R.T., overnight; (xii) H₂, 10% Pd/C, EtOH, R.T. overnight;(xiii) TMSBr, CH₂Cl₂, R.T., 4 h., then 95% CH₃OH in H₂O, R.T., 1 h.

2-Bromo-1-(4-octyl-phenyl)-ethanone (1). To a flame dried round bottomflask equipped with a magnetic stirbar under an inert atmosphere wasadded AlCl₃ (5.47 g; 41 mmol) followed by 1,2-dichloroethane (22 mL).The stirring suspension was then brought to 0° C. and 1-phenyloctane(7.99 mL, 36 mmol) was added in one portion. Bromoacetyl bromide (3.75mL, 43 mmol) was then added dropwise over a period of 10 minutes. Uponcompleting addition of the acid bromide, the reaction mixture wasbrought to rt and stirred for 2 h. The reaction mixture was thenquenched carefully by slow addition of H₂O (36 mL) without ever lettingthe reaction mixture exceed 45° C. producing a suspension of solid whiteprecipitate. The aqueous layer of the quenched reaction mixture wasdiscarded and the organic phase washed once with 10% HCl (10 mL), washedonce with H₂O (10 mL), and dried over magnesium sulfate. The driedorganic phase was then concentrated in vacuo to a green/brown oil.Recrystallization from MeOH/H₂O provided the product 1 (6.36 g, 57%) aswhite needles in three crops. R_(f)=0.21 (1:19 EtOAc/hexanes).

2-Amino-3-hydroxy-2-methyl-propionic acid methyl ester (2). A stirringsolution of α-methyl-DL-Serine (1 g, 8.39 mmol) in MeOH (40 mL) in aflame dried round bottom flask under an inert atmosphere was cooled to0° C. and SOCl₂ (1.84 mL, 25.19 mmol) was slowly added. After additionof the SOCl₂ was complete, the reaction mixture was stirred 12 h at rtand then concentrated in vacuo to a white solid that was used directlyin the next reaction.

2-tert-Butoxycarbonylamino-3-hydroxy-2-methyl-propionic acid methylester (3). To the crude product obtained in the above reaction wasslowly added sat. aq. NaHCO₃ (12.5 mL) followed by solid NaHCO₃ (500 mg)and the reaction mixture was stirred 30 min under an inert atmosphere.THF (12.5 mL) was then added to the reaction mixture followed bydi-tert-butyl dicarbonate (1.83 g, 8.39 mmol) and stirring at rt wascontinued for 12 h. The reaction mixture was then diluted with H₂O (20mL) and extracted with EtOAc (3×20 mL). The combined EtOAc extracts weredried over sodium sulfate and concentrated in vacuo to a thick whitepaste. To this paste was added hexanes which produced 3 (630 mg, 32% for2 steps) as a white precipitate which was collected by filtration.R_(f)=0.35 (1:1 EtOAc/hexanes).

2,2,4-Trimethyl-oxazolidine-3,4-dicarboxylic acid 3-tert-butyl ester4-methyl ester (4). To a stirring solution of 3 (9.342 g, 40 mmol) inacetone (115 mL) in a flame dried round bottom flask under an inertatmosphere was added 2,2-dimethoxypropane (66 mL). To this solution wasadded BF₃.OEt₂ (0.30 mL, cat.) and stirring was continued at rt for 2 h.The reaction mixture was then concentrated in vacuo to an orange oilwhich was purified by flash chromatography to provide 4 (9.392 g, 85%)as a white solid. R_(f)=0.55 (1:3 EtOAc/hexanes). Compound was observedas an uneven mixture of rotomers.

2,2,4-Trimethyl-oxazolidine-3,4-dicarboxylic acid 3-tert-butyl ester(5). To a stirring solution of 4 (9.392 g, 34 mmol) in THF (65 mL) andH₂O (35 mL) under an inert atmosphere was added solid LiOH.H₂O (1.426 g,34 mmol) in one portion. The reaction mixture was heated to 90° C. andstirred 8 h at which point the reaction mixture was cooled to rt. Thecrude reaction mixture washed with Et₂O (3×50 mL) and the Et₂O extractswere discarded. The aqueous solution was then acidified with 2M KHSO₄until a white precipitate began to form on addition, pH=5. The acid wasadded dropwise until the precipitate persisted and the aqueous solutionwas extracted with Et₂O (50 mL). After extraction, two addition drops ofacid were added to the aqueous layer and it was again extracted withEt₂O (25 mL). The Et₂O extracts were combined and quickly back extractedwith 1M NaOH (15 mL). The organic phase was then dried over sodiumsulfate and concentrated in vacuo to give 5 (7.458 g, 85%) as a whitesolid which was used without further purification. Compound was observedas an uneven mixture of rotomers.

2,2,4-Trimethyl-4-[5-(4-octyl-phenyl)-1H-imidazol-2-yl]-oxazolidine-3-carboxylicacid tert-butyl ester (6). To a flame dried round bottom flask equippedwith a magnetic stirbar under an inert atmosphere was added 5 (3.00 g,11.6 mmol) followed by absolute EtOH (33 mL) and Cs₂CO₃ (1.93 g, 5.9mmol). This mixture was then shaken 30 min at which time all of thesuspended Cs₂CO₃ had disappeared. The reaction mixture was thenconcentrated in vacuo to a white solid at which time DMF (60 mL) wasadded. To the stirring solution was added a solution of 1 (3.60 g, 11.6mmol) in DMF (5 mL). The resulting solution was stirred 4 h andconcentrated to a light brown solid. To the light brown solid was addedEtOAc (50 mL) and the suspended CsBr was filtered off and washed withEtOAc. The filtrate was then concentrated to a light brown foam whichwas subsequently dissolved in xylenes (195 mL) in a round bottom flaskequipped with a Dean-Stark trap (filled with xylenes) and a refluxcondenser. To this solution was added NH₄OAc (1.74 g, 22.6 mmol) and thereaction mixture was brought to 105° C. and stirred 3 h at which timethe reaction would progress no further. The crude reaction mixture wasthen concentrated in vacuo to a red oil. To the oil was added EtOAc (200mL) and this solution washed with sat. aq. NaHCO₃ (3×50 mL) followed bybrine solution (1×50 mL). The organic phase was then dried over sodiumsulfate and concentrated to a red oil which was subjected to flashchromatography to give 6 (1.074 g, 20%) as a white solid. R_(f)=0.45(6:4 Et₂O/petroleum ether).

2-Amino-2-[5-(4-octyl-phenyl)-1H-imidazol-2-yl]-propan-1-ol (VPC24241).To a flame dried round bottom flask equipped with a magnetic stirbarunder an inert atmosphere was added 6 (973 mg, 2.07 mmol) followed byMeOH (20 mL) and p-TsOH.H₂O (1.22 g, 6.42 mmol). This mixture was thenheated to reflux, stirred 3 h, cooled to 0° C., and quenched by slowaddition of sat. aq. NaHCO₃ (20 mL). This solution was then diluted withEtOAc (30 mL) and the aqueous layer was discarded. The organic phasewashed with sat. aq. NaHCO₃ (1×20 mL), washed with 1M NaOH (1×20 mL),dried over sodium sulfate, and concentrated to an orange oil. To thisoil was added Et₂O which produced VPC24241 (408 mg, 60%) as a whiteprecipitate which was collected by filtration.

{2-Hydroxy-1-methyl-1-[5-(4-octyl-phenyl)-1H-imidazol-2-yl]-ethyl}-carbamicacid tert-butyl ester (7). To a vigorously stirring solution of VPC24241(70 mg, 0.213 mmol) in THF (4 mL) and H₂O (2 mL) was added Na₂CO₃ (198mg, 1.87 mmol) followed by di-tert-butyl dicarbonate (214 mg, 0.98 mmol)and the resulting solution was stirred 12 h at rt. The reaction mixturewas then diluted with EtOAc (20 mL) and washed with saturated aq. NaHCO₃(2×15 mL). The organic phase was dried over sodium sulfate andconcentrated in vacuo to a clear oil which solidified to a white solidunder vacuum. This white solid was then subjected to flashchromatography to produce 7 (52 mg, 57%) as a white solid. R_(f)=0.50(1:1 EtOAc/hexanes).

{2-(Di-tert-butoxy-phosphoryloxy)-1-methyl-1-[5-(4-octyl-phenyl)-1H-imidazol-2-yl]-ethyl}-carbamicacid tert-butyl ester (8). To a solution of 7 (33 mg, 0.077 mmol) in 1:1CH₂Cl₂/THF (3 mL) was added a 3% solution of tetrazole in acetonitrile(0.44 mL, 0.154 mmol) and the resulting mixture was stirred 30 min.Di-tert-butyl-di-isopropylphosphoramidite (0.05 mL, 0.154 mmol) was thenadded and the resulting reaction mixture was stirred 12 h. To thissolution was added 30% hydrogen peroxide (0.04 mL, 0.308 mmol) and theresulting mixture was stirred 3 h, cooled to 0° C., and quenched byaddition of aqueous Na₂S₂O₅. The resulting solution was diluted withethyl acetate (10 mL) and washed with saturated aq. NaHCO₃ (2×5 mL). Theorganic layer was dried over sodium sulfate and the solvent was removedunder reduced pressure to afford a clear oil. Flash chromatography,using 1:1 EtOAc/hexanes, provided 8 (22 mg, 46%) as a clear oil.R_(f)=0.45 (1:1 EtOAc/hexanes).

{2-(Di-tert-butoxy-thiophosphoryloxy)-1-methyl-1-[5-(4-octyl-phenyl)-1H-imidazol-2-yl]-ethyl}-carbamicacid tert-butyl ester (9). To a solution of 7 (19 mg, 0.044 mmol) in 1:1CH₂Cl₂/THF (2 mL) was added a 3% solution of tetrazole in acetonitrile(0.26 mL, 0.089 mmol) and the resulting mixture was stirred 30 min.Di-tert-butyl-di-isopropylphosphoramidite (0.03 mL, 0.089 mmol) was thenadded and the resulting reaction mixture was stirred 12 h. To thissolution was added elemental sulfur (excess) and the resulting mixturewas stirred 12 h. The resulting solution was diluted with ethyl acetate(7 mL) and washed with saturated aq. NaHCO₃ (2×3 mL). The organic layerwas dried over sodium sulfate and the solvent was removed under reducedpressure to afford a clear oil with yellow tint. Flash chromatography,using 1:3 EtOAc/hexanes, provided 9 (13 mg, 46%) as a clear oil.R_(f)=0.40 (1:3 EtOAc/hexanes).

Phosphoric acidmono-{2-amino-2-[5-(4-octyl-phenyl)-1H-imidazol-2-yl]-propyl}ester(VPC24287). To a stirring solution of 8 (22 mg, 0.035 mmol) in CH₂Cl₂ (1mL) was added trifluoroacetic acid (1 mL) and stirring was continued 4h. Solvent and excess trifluoroacetic acid were removed in vacuo toafford a brown oil. The oil was diluted with ether and concentrated invacuo 5 times on a rotary evaporator to afford a white solid which wasplaced in a fritted funnel and washed with cold ether producing VPC24287(13 mg, 91%) as a powdery white solid. R_(f)=0 (4:1 CHCl₃/methanol).

Thiophosphoric acidO-{2-amino-2-[5-(4-octyl-phenyl)-1H-imidazol-2-yl]-propyl}ester(VPC24289). To a stirring solution of 9 (13 mg, 0.020 mmol) in CH₂Cl₂ (1mL) was added benzenethiol (0.042 mL, 0.40 mmol) followed bybromotrimethyl silane (0.05 mL, 0.40 mmol) and finally trifluoroaceticacid (1 mL) and stirring was continued 6 h. To quench the reactionmixture, water (0.5 mL) was added and the resulting solution was stirred30 min. Solvent and excess reagents were removed in vacuo to afford abrown oil. The oil was diluted with ether and concentrated in vacuo 5times on a rotary evaporator to afford a light tan solid which wasplaced in a fritted funnel and washed with cold ether and a small amountof cold water producing VPC24289 (8 mg, 94%) as a powdery white solid.R_(f)=0 (4:1 CHCl₃/metha.

Synthetic Scheme for Synthesis of Additional Imidizole Compounds

Reagents and Conditions: (i) Br₂, 1:1 dioxane/ether, CH₂Cl₂, rt, 1 h,66%; (ii) 2,2-DMP, p-TsOH, DMF, rt, 12 h, TEA, rt, 10 min; (iii)(Boc)₂O, NaHCO₃, THF/H₂O, rt, 12 h, 69% (2 steps); (iv) (COCl)₂, DMSO,TEA, CH₂Cl₂, −78° C. to rt, 4 h, 74%; (v) NaClO₂, NaH₂PO₄.H₂O,2-methyl-2-butene, ^(t)BuOH/H₂O, rt, 1 h, 95%; (vi) Cs₂CO₃, EtOH, rt, 1h; 1, DMF, rt, 12 h; (vii) NH₄OAc, xylenes, 110° C., 12 h, 36% (2steps); (viii) Pd(dba)₂, Ph₃P, CuI, DIEA, THF, rt, 12 h, 68%; (ix) H₂,10% Pd/C, EtOH, rt, 12 h; (x) 1:1 TFA/CH₂Cl₂, rt, 6 h; (xi) DOWEX 50×8,EtOH, rt, 12 h; (xii) tetrazole, di-tert-butyldiisopropylphosphoramidite, CH₂Cl₂/THF, rt, 12 h; H₂O₂, rt, 3 h; (xiii)tetrazole, di-tert-butyl diisopropylphosphoramidite, CH₂Cl₂/THF, rt, 12h; S₈, rt, 3 h; (xiv) 1:1 TFA/CH₂Cl₂, rt, 4 h; (xv) benzenethiol, TMSBr,1:1 TFA/CH₂Cl₂, rt, 4 h.

Synthetic Scheme for Synthesis of Alpha Substituted PhosphonateCompounds

Reagents and Conditions: (i) NaH, THF 0° to R.T. 45 min., thenSelectfluor 0° to R.T., overnight, 53%; (ii) SOCl₂, MeOH, R.T., 4-6 h.;(iii) Boc₂O, TEA, CH₂Cl₂, R.T., 4 h.; (iv) 2,2-dimethoxypropane,p-toluenesulfonic acid, CH₂Cl₂, R.T., 2 h., 62% (3 steps) (v) LiCl,NaBH₄, EtOH/THF (3:2), 0° to R.T., 4 h, 89%; (vi) PCC, CH₂Cl₂, R.T., 6h.; (vii) DBU, LiCl, CH₃CN, R.T., overnight, 40% (2 steps); (viii) Dowex50×8, EtOH, R.T. 24 h., 80%; (ix) PCC, CH₂Cl₂, R.T., 6 h. (x) NaClO₂,NaH₂PO₄.H₂O, t-butanol, 2-methyl-2-butene; (xi) p-octyl aniline, PyBOP,DIEA, CH₂Cl₂, R.T., overnight; (xii) H₂, 10% Pd/C, EtOH, R.T. overnight;(xiii) TMSBr, CH₂Cl₂, R.T., 4 h., then 95% CH₃OH in H₂O, R.T., 1 h.

[(Diethoxy-phosphoryl)-fluoro-methyl]-phosphonic acid diethyl ester(31). To a slurry of 95% NaH (9 mg, 0.375 mmol) in THF (1.5 mL) wasadded tetraethyl methylene diphosphonate, (30) (100 mg, 0.347 mmol) at0° C. The mixture was allowed to warm to room temperature and stirredfor 45 minutes. The mixture was subsequently cooled to 0° C. andSelectfluor (153 mg, 0.432 mmol) was added in one portion. The mixturewas allowed to warm to room temperature and stirred for 1 hr. Thereaction mixture was concentrated in vacuo and purified by columnchromatography on SiO₂ (3% MeOH in EtOAc) to yield 56 mg (53%) of aclear liquid.

2-Amino-3-hydroxy-propionic acid methyl ester (33). To a solution ofD-serine (5 g, 47.58 mmol) in methanol (100 mL), stirring underN_(2 (g)) at 0° C., was added thionyl chloride (20.8 mL, 285.5 mmol)dropwise. The reaction mixture was allowed to warm to room temperature,stirred for 4-6 hours, then concentrated under reduced pressure. Thecrude material was reconstituted in Et₂O and concentrated, in the samemanner. This was repeated numerous times until SOCl₂ could not bedetected. The crude material was confirmed by NMR experiments andcarried on to the following step.

2-tert-Butoxycarbonylamino-3-hydroxy-propionic acid methyl ester (34).To a solution of the crude methyl ester serine (33) in CH₂Cl₂ (100 mL),stirring under N_(2 (g)), was added di-tert-butyl pyrocarbonate (11.420g, 52.34 mmol) and triethyl amine (16.6 mL, 118.95 mmol). The reactionmixture was allowed to stir at room temperature for 4 hours, then pouredover NH₄Cl at 0° C. The organic layer was extracted with 10% HCl (2×),then NaHCO₃ and brine. The organic layer was then dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure. The crudematerial was again carried on to the following step.

2,2-Dimethyl-oxazolidine-3,4-dicarboxylic acid 3-tert-butyl ester4-methyl ester (35). To a solution of (34) in CH₂Cl₂, stirring undernitrogen at 0° C., was added 2,2-dimethoxypropane (29.5 mL, 237.9 mmol)and p-toluene sulfonic acid monohydrate (9.050 g, 47.58 mmol). Themixture was removed from the ice bath after 15 minutes and stirred atroom temperature for 1.5 hours. The reaction mixture was poured into 50mL of saturated NaHCO_(3 (aq)) and extracted with diethyl ether (3×50).The organic layer was extracted with NaHCO₃ and brine, then dried overanhydrous MgSO₄, filtered and concentrated under reduced pressure. Thecrude material was purified by flash chromatography on SiO₂ (1:1EtOAc/Hexanes) to yield 7.659 g (62%, 3 steps) of a clear liquid.

4-Hydroxymethyl-2,2-dimethyl-oxazolidine-3-carboxylic acid tert-butylester (36). To a mixture of NaBH₄ (2.247 g, 59.08 mmol) and LiCl (2.505g, 59.08 mmol) in EtOH (42 mL), stirring under nitrogen at 0° C., wasadded (35) (7.659 g, 29.54 mmol) in THF (30 mL) dropwise. This mixturewas allowed to warn to room temperature and continued stirring for 48hours. The precipitate was filtered and washed with ethanol. Thewashings were concentrated and extracted with EtOAc. The organic layerwas then washed with brine and dried over anhydrous Na₂SO₄. Columnchromatography on SiO₂ (1:1 EtOAc/Hexanes) was utilized to purify 6.101g (89%) of the title compound as a white solid.

4-Formyl-2,2-dimethyl-oxazolidine-3-carboxylic acid tert-butyl ester(37). To a solution of (36) (80 mg, 0.346 mmol) stirring in CH₂Cl₂ (2mL), under a nitrogen atmosphere, was added pyridinium chlorochromate(150 mg, 0.694 mmol). The reaction mixture was allowed to stir overnightthen filtered through a plug of silica gel. The crude aldehyde wascarried on to the following step.

4-[2-(Diethoxy-phosphoryl)-2-fluoro-vinyl]-2,2-dimethyl-oxazolidine-3-carboxylicacid tert-butyl ester (38). To a stirred suspension of LiCl (18 mg,0.416 mmol) in dry acetonitrile (3.5 mL), under nitrogen at roomtemperature, were added diphosphonate (31) (127 mg, 0.416 mmol), DBU(0.05 mL, 0.347 mmol) and garner's aldehyde (37) (80 mg, 0.347 mmol).The reaction mixture was allowed to stir overnight then concentrated invacuo. The crude material was isolated by column chromatography on SiO₂,(1:1 EtOAc/Hexanes) to yield 47 mg (40%, two steps) of a clear liquid.

(3-tert-Butoxycarbonylamino-1-fluoro-4-hydroxy-but-1-enyl)phosphonicacid diethyl ester (39). To compound (38) (47 mg, 0.123 mmol) stirringin EtOH (1 mL) was added Dowex 50×8 (150 mg), which washed with EtOH anddried. The reaction was allowed to stir under nitrogen and at roomtemperature for 24 hours. The reaction mixture was filtered and theprecipitate washed with excess EtOH, then concentrated in vacuo. Thecrude material was purified by column chromatography on SiO₂ (1:1EtOAc/Hexanes) to yield 34 mg of the expected product.

EXAMPLE 3

[γ-35 S]GTP Binding Assay for Measuring S1P Activity

Transient Expression in HEK293T Cells.

Human or mouse S1P5 DNA was mixed with an equal amount of DNA encoding arat Gi2R protein as well as DNAs encoding cow β1 and γ2 proteins andused to transfect monolayers of HEK293T cells using the calciumphosphate precipitate method. After 60 h, cells were harvested, andmicrosomes were prepared, aliquoted, and stored at −70° C. until use.

[γ-35 S]GTP Binding.

Briefly, 5 ug of membranes from S1P expressing HEK293T cells wasincubated in 0.1 mL of binding buffer (in mM: HEPES 50, NaCl 100, MgCl₂5), pH 7.5, containing 5 ug of saponin, 10 uM GDP, 0.1 nM [γ-35 S]GTP(1200 Ci/mmol), and test lipid. After incubating for 30 min at 30° C.,bound radionuclide was separated from free by filtration through WhatmanGF/C paper using a Brandel Cell Harvester (Gaithersburg, Md.).

Stable Expression in RH7777 Cells.

Rat hepatoma RH7777 cell monolayers were transfected with human or mouseS1P5/pCR3.1 DNA using the calcium phosphate precipitate method, andclonal populations expressing the neomycin phosphotransferase gene wereselected by addition of Ge-neticin (G418) to the culture medium. TheRH7777 cells were grown in monolayers at 37° C. in a 5% CO₂/95% airatmosphere in growth medium consisting of 90% MEM, 10% fetal bovineserum, 2 mM glutamine, and 1 mM sodium pyruvate.

Measurement of cAMP Accumulation.

Assay of cAMP accumulation was performed as described previously (See Imet al., J. Biol. Chem. 275, 14281-14286 (2000), the disclosure of whichis incorporated herein). Assays were conducted on populations of 5×10⁵cells stimulated with 1 uM forskolin in the presence of thephosphodiesterase inhibitor isomethylbutylxanthine (IBMX, 1 mM) for 15min. cAMP was measured by automated radioimmunoassay. The GTPγS studieswere performed using zebrafish S1P1 overexpressed rat RH-7777 and humanhS1P1, hS1P2, hS1P3 and hS1P5 overexpressed human HEK293 cells. Table 1shows the EC₅₀ values for each of the S1P analogs at S1P receptors:S1P1, S1P2, S1P3 and S1P5. In addition to testing the human S1Preceptors (hS1P1, hS1P2, hS1P3 and hS1P5), a zebrafish S1P receptor(zS1P1) and mouse S1P (mS1P5) were also tested.

TABLE 1 EC₅₀ Values (nM) for S1P Analogues at Recombinant S1P ReceptorszS1P₁ hS1P₁ hS1P₃ hS1P₂ hS1P₅ mS1P₅ S1P 54.6 0.9 1.1 2.9 43.9 12.7VPC22041 2053.0 598.4 845.4 973.2 645.5 >5000 VPC22051 >5000 322.1 601.92760.0 >5000 >5000 VPC22053 >5000 397.0 862.4 2685.0 1606.0 2006.0VPC22063 >5000 1805.0 878.6 >5000 1220.0 1326.0 VPC22135 1625.0 12.750.8 2107.0 >5000 1821.0S1P increases GTPγS binding significantly (2-5-fold) at each receptorwith EC₅₀ values from 1 to 55 nM. The synthetic series consisted of fivedihydro S1P of the general formula:

wherein

-   -   VPC22041 (2S): R₁ is NH(CH₂)₁₁CH₃, R₂ is NH₂ and R₃ is H;    -   VPC22053 (2S): R₁ is O(CH₂)₁₃CH₃, R₂ is NH₂ and R₃ is H;    -   VPC22051 (2S): R₁ is NH(CH₂)₁₃CH₃, R₂ is NH₂ and R₃ is H;    -   VPC22063 (2S): R₁ is NH(CH₂)₁₅CH₃, R₂ is NH₂ and R₃ is H; and    -   VPC22135 (2R): R₁ is NH(CH₂)₁₃CH₃, R₂ is H and R₃ is NH₂

The amide-containing compounds contained alkyl chains of 12 (VPC22041),14 (VPC22053), or 16 (VPC22063) carbons, and the 2′-amino group was inthe natural configuration (S), except for VPC22135, wherein the 2′-aminowas in the (R) configuration. VPC22053 and VPC22135 are an enantiomericpair, while VPC22051 is the ester-containing equivalent of VPC22053 (seeScheme 4). All of these compounds had significant agonist activity ateach of the S1P receptors, although none were as potent as S1P itself(see Table 1). In particular, on the S1P5 transfected HEK293 cells, thefive mimetics showed EC₅₀=s of approximately 1 μM, where as the EC₅₀ ofS1P itself on the same cells is closer to 10 nM. However, one compound,VPC22135, approached the potency of S1P at both the human S1P1 and humanS1P3 receptors. Curiously, this compound has the amino group in theunnatural (R) configuration. Its enantiomer, VPC22053, was more than 1log order less potent at both the S1P1 and S1P3 receptors. The resultsobtained for the S1P1 transfected RH-7777 cells showed a preference forbinding with the 18 carbon backbone mimetic compounds (identical to S1P)over the 16 and 20 carbon backbone mimetic compounds.

Assay of phenyl imidazole compounds vpc24287 (phosphate) and vpc24289(phosphothionate) at individual human sphingosine 1-phosphate (S1P)receptors was also conducted.

Methods: Human recombinant S1P receptor type DNAs were mixed with DNAsencoding human Gαi2, cow β1 and cow 72 proteins and introduced intocultured HEK293T cells by transfection. After about 48 hours, cells wereharvested and crude membranes prepared. Ligand driven binding of anon-hydrolyzable GTP analog, GTP[γ-³⁵S], was measured in a rapidfiltration assay. Details of the assay are found in: Brinkmann, V.,Davis, M. D., Heise, C. E., Albert, R., Cottens, W., H of, R., Bruns,C., Prieschl, E., Baumruker, T., Hiestand, P., Foster, C. and Lynch, K.R. The immune modulator, FTY720, targets sphingosine 1-phosphatereceptors. J. Biol. Chem. 277: 21453-21457 (2002). Total counts perminute were determined for S1P, vpc24287 and vpc24289 activation of theS1P receptor subtypes with the maximal counts received by S1P designatedas 100% activation of the S1P receptor. The results are provided in FIG.6A-6D demonstrating vpc24287 and vpc24289 activation of the S1P receptorsubtypes relative to S1P.

EXAMPLE 4

Biological Assay of the Synthesized Mimetics

An additional series of compounds was tested using the GTP_(C)S bindingassay described in Example 2 and in Im et al., J. Biol. Chem. 275,14281-14286 (2000), the disclosure of which is incorporated herein). Thecompounds tested for binding at human S1P receptors (hS1P1, hS1P2,hS1P3, hS1P4 and hS1P5) have the general structure:

wherein

-   -   VPC23019: R₅ is (CH₂)₇CH₃, R₂ is NH₂, R₃ is H and R₄ is        phosphate;    -   VPC23031: R₅ is (CH₂)₇CH₃, R₂ is NH₂, R₃ is H and R₄ is        phosphate;    -   VPC23065: R₅ is (CH₂)₉CH₃, R₂ is NH₂, R₃ is H and R₄ is hydroxy;    -   VPC23069: R₅ is (CH₂)₉CH₃, R₂ is NH₂, R₃ is H and R₄ is        phosphate;    -   VPC23075: R₅ is (CH₂)₈CH₃, R₂ is NH₂, R₃ is H and R₄ is hydroxy;    -   VPC23079: R₅ is (CH₂)₈CH₃, R₂ is NH₂, R₃ is H and R₄ is        phosphate;    -   or have the general structure:

wherein

-   -   VPC23087: R₅ is (CH₂)₇CH₃, R₂ is NH₂, R₃ is H and R₄ is hydroxy;    -   VPC23089: R₅ is (CH₂)₇CH₃, R₂ is NH₂, R₃ is H and R₄ is        phosphate;

Each of the compounds tested (VPC 23019, 23031, 23065, 23069, 23087,23089, 23075, 23079) failed to show significant activity at the S1P2receptor.

Compounds VPC23065, VPC23087 and VPC23075 are primary alcohols and thuslack the phosphate headgroup. Yet several of these compounds exhibitactivity at S1P receptors (See FIGS. 2A, 2B, 2C, 3A, 3B, 3C and 4C) andeach of these compounds shows good agonist activity at the S1P4receptor.

The GTP_(C)S binding assay revealed that VPC23031, VPC23019, VPC23089are inverse agonists (antagonists) of the S1P3 receptor (See FIGS. 1Aand 4A), but this inverse agonism becomes agonism when the alkyl chainlength is 9 carbons (VPC23079) or 10 (VPC23069), see FIGS. 2A and 3A.

VPC23089 and VPC23019 are isomers, with the VPC23089 compound having thealkyl chain ortho and the VPC23019 compound meta; in both cases thealkyl chain has 8 carbons, but surprisingly, when one goes from ortho tometa, antagonism at S1P1 is realized (compare FIG. 1A with thecompetition curve FIG. 5A).

1. A compound of the formula:

wherein R₁₁ is C₅-C₁₂ alkyl, C₅-C₁₂ alkenyl, C₅-C₁₂ alkynyl, C₅-C₁₂alkoxy, C₅-C₁₀ (aryl)R₂₀, C₅-C₁₀ (heteroaryl)R₂₀, C₅-C₁₀(cycloalkyl)R₂₀, C₅-C₁₀ alkoxy(aryl)R₂₀, C₅-C₁₀ alkoxy(heteroaryl)R₂₀ orC₅-C₁₀ alkoxy(cycloalkyl)R₂₀; wherein R₂₀ is H or C₁-C₁₀ alkyl; R₂₉ is Hor halo; R₂ is NH₂; R₃ is H, C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, or (C₁-C₄alkyl)NH₂; R₂₃ is H, F, NH₂, OH, CO₂H, C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, or(C₁-C₄ alkyl)NH₂; R₂₄ is H, F, C₁-C₄ alkyl or PO₃H₂; or R₂₃ togetherwith R₂₄ and the carbon to which they are attached form a carbonylgroup; R₂₅, R₇, and R₈ are independently O, S, CHR₂₆, CR₂₆, NR₂₆, or N;wherein R₂₆ is H, F or C₁-C₄ alkyl; R₁₅ is hydroxy, phosphonate, or

wherein R₁₂ is O, NH or S; X is O, NH or S; y and m are integersindependently ranging from 0 to 4; or a pharmaceutically acceptable saltor tautomer thereof.
 2. The compound of claim 1, of the formula:

wherein R₁₁ is C₅-C₁₂ alkyl, C₅-C₁₂ alkenyl, C₅-C₁₂ alkoxy, or C₅-C₁₂alkynyl; R₇ and R₈ are independently O, S, CHR₂₆, CHR₂₆NR₂₆, or N;wherein R₂₆ is H, F or C₁-C₄ alkyl; R₂₅ is N or CH; R₂ is NH₂; R₃ is H,C₁-C₄ alkyl, (C₁-C₄ alkyl)OH, or (C₁-C₄ alkyl)NH₂; R₁₅ is hydroxy,phosphonate, or

wherein X and R₁₂ are independently O or S; R₂₃ is H, F, OH, C₁-C₄alkyl, CO₂H or (C₁-C₄ alkyl)OH; R₂₄ is H, F, C₁-C₄ alkyl or PO₃H₂, orR₂₃ together with R₂₄ and the carbon to which they are attached form acarbonyl group; and y and m are integers independently ranging from 0 to4; or a pharmaceutically acceptable salt or tautomer thereof.
 3. Thecompound of claim 2 wherein m is 0; y is 0 or 1; R₂₃ is H or F; and R₂₄is H, F or C₁-C₄ alkyl; or a pharmaceutically acceptable salt ortautomer thereof.
 4. The compound of claim 2, wherein R₃ is C₁-C₃ alkylor (C₁-C₄ alkyl)OH; or a pharmaceutically acceptable salt or tautomerthereof.
 5. The compound of claim 3, wherein R₇ is NH; and X is O; or apharmaceutically acceptable salt or tautomer thereof.
 6. The compound ofclaim 5, wherein y is 0; and R₁₅ is OH; or a pharmaceutically acceptablesalt or tautomer thereof.
 7. The compound of claim 4, of the formula:

wherein R₁₁ is C₅-C₁₂ alkyl, C₅-C₁₂ alkoxy, or C₅-C₁₂ alkenyl; and R₈ isN; or a pharmaceutically acceptable salt or tautomer thereof.
 8. Thecompound of claim 7, wherein R₁₅ is hydroxy or

wherein R₁₂ is O or S; or a pharmaceutically acceptable salt or tautomerthereof.
 9. The compound of claim 8, wherein R₁₁ is C₅-C₉ alkyl; R₁₅ isOH and R₃ is CH₃, CH₂CH₃, CH₂OH, CH₂CH₂OH or CH₂CH₂CH₂OH; or apharmaceutically acceptable salt or tautomer thereof.
 10. The compoundof claim 7, 2-amino-2-[5-(4-octyl-phenyl)-1H-imidazol-2-yl]-propan-1-ol;or a pharmaceutically acceptable salt or tautomer thereof.
 11. Acomposition comprising a compound of claim 1, and a pharmaceuticallyacceptable carrier.
 12. A pharmaceutical composition comprising acompound of the formula

wherein R₁₁ is C₅-C₁₈ alkyl, C₅-C₁₈ alkoxy, or C₅-C₁₈ alkenyl; Q isC₃-C₆ optionally substituted cycloalkyl, C₃-C₆ optionally substitutedheterocyclic, C₃-C₆ optionally substituted aryl, C₃-C₆ optionallysubstituted heteroaryl or —NH(CO)—; R₃ is H, C₁-C₄ alkyl or (C₁-C₄alkyl)OH; R₂₃ is H or C₁-C₄ alkyl, and R₁₅ is hydroxy, phosphonate, or

wherein X and R₁₂ is O or S; or a pharmaceutically acceptable salt ortautomer thereof and a pharmaceutically acceptable carrier.
 13. Thecomposition of claim 12 wherein Q is


14. The composition of claim 13 wherein R₁₅ is hydroxy or

wherein R₁₂ is O or S.
 15. The composition of claim 14 wherein Q is

R₁₅ is OH; or a pharmaceutically acceptable salt or tautomer thereof.